Piv5 as an oncolytic agent

ABSTRACT

The present invention includes the  Paramyxovirus  Parainfluenza Virus 5 (PIV5) as an oncolytic agent for treating various cancers, including, but not limited to breast cancer, lung cancer and melanoma. PIV5 oncolytic agents include both wild type PIV5 and various recombinant PIV5 constructs. Recombinant PIV5 constructs may include PIV5 lacking the conserved C-terminus of the V protein (PIV5VΔC), PIV5 with mutations in the N-terminus of the V/P protein (PIV5CPI−), and PIV5 expressing MDA-7/IL-24 (rPIV5-MDA7), rPIV5-V/P-CPI−, rPIV5-CPI+, rPIV5-Rev, rPIV5-RL, rPIV5-P-S157A, rPIV5-P-S308A, rPIV5-L-A1981D, rPIV5-F-S443P, rPIV5-MDA7, rPIV5ΔSH-CPI−, or rPIV5ΔSH-Rev. Also included are methods of making and using such oncolytic agents and compositions including such oncolytic agents.

CONTINUING APPLICATION DATA

This application is a continuation of U.S. application Ser. No.15/638,946, filed Jun. 30, 2017, which is a divisional of U.S. NationalStage Application No. 14/374,070, filed Jul. 23, 2014, which is the §371 U.S. National Stage of International Application No.PCT/US2013/022898, filed 24 Jan. 2013, which claims the benefit of U.S.Provisional Applications Ser. Nos. 61/590,056, filed Jan. 24, 2012,61/590,070, filed Jan. 24, 2012, and 61/683,810, filed Aug. 16, 2012,each of which is incorporated by reference herein.

GOVERNMENT FUNDING

This invention was made with government support under Grant No.R01AI070847 and R56A1081816, awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

BACKGROUND

Cancer causes significant morbidity and mortality in human populations.Surgery, chemotherapy, and irradiation have been used to treat andcontrol cancers. However, these approaches are not effective againstmany late stage cancers. In addition, chemotherapy and irradiation oftenhas severe unwanted side effects. For example, it is estimated that over230,000 new cases of invasive breast cancer will be diagnosed in womenin the United States in 2011. Another nearly 40,000 will die from thedisease. Breast cancer is second only to lung cancer in causing cancerrelated deaths in women. This grim outlook seems to contrast the factthat breast cancer detected early is very curable. However, once itmetastasizes the cure rate drops precipitously. The standard of care,surgery and chemotherapy, often do not prevent future metastasis even inpatients who have been pronounced cured. Thus, there is a need for novelstrategies for cancer control, including the treatment of late stagecancers and cancer metastasizes.

SUMMARY OF THE INVENTION

The present invention includes a method of killing a tumor cell,reducing the growth of a tumor cell, reducing tumor size, inducing tumorcells syncytia formation, and/or inducing apoptosis in a tumor cell, themethod including infecting the tumor cell with a composition includingan isolated a recombinant PIV5 (rPIV5).

The present invention includes a method of killing tumor cells in asubject, the method including administering to the subject an effectiveamount of a composition including an isolated recombinant parainfluenzavirus 5 (PIV5).

The present invention includes a method of treating a subject with acancer, the method including administering to the subject an effectiveamount of a composition including an isolated recombinant parainfluenzavirus 5 (PIV5).

The present invention includes a method of imaging a tumor in a subject,the method including administering to the subject a recombinantparainfluenza virus 5 (PIV5) expressing a fluorescent polypeptide ordetectable agent.

In some embodiments of the methods described herein, the subject is acompanion animal. In some embodiments, the companion animal is a dog.

In some embodiments of the methods described herein, the tumor is aprimary tumor and/or a metastatic tumor.

In some embodiments of the methods described herein, the tumor ismelanoma, basal cell carcinoma, colorectal cancer, pancreatic cancer,breast cancer, prostate cancer, lung cancer (including small-cell lungcarcinoma and non-small-cell lung carcinoma, leukemia, lymphoma,sarcoma, ovarian cancer, Kaposi's sarcoma, Hodgkin's lymphoma,Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, head andneck cancers, malignant pancreatic insulanoma, malignant carcinoid,urinary bladder cancer, premalignant skin lesions, testicular cancer,lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, cervical cancer,kidney cancer, endometrial cancer, glioblastoma, or adrenal corticalcancer.

In some embodiments of the methods described herein, administration ofPIV5 is intratumoral, subcutaneous, intravenous, intranasal,intraperitoneal, intracranial, oral, or in situ.

In some embodiments of the methods described herein, the method furtherincludes administration of an additional therapeutic agent.

In some embodiments of the methods described herein, the PIV5 includesone or more mutations. In some embodiments, a mutation includes amutation of the V/P gene, a mutation of the shared N-terminus of the Vand P proteins, a mutation of residues 26, 32, 33, 50, 102, and/or 157of the shared N-terminus of the V and P proteins, a mutation lacking theC-terminus of the V protein, a mutation lacking the small hydrophobic(SH) protein, a mutation of the fusion (F) protein, a mutation of thephosphoprotein (P), a mutation of the large RNA polymerase (L) protein,a mutation incorporating residues from canine parainfluenza virus,and/or a mutation that enhances synctial formation. In some embodiments,a mutation is selected from the group consisting of rPIV5-V/P-CPI−,rPIV5-CPI−, rPIV5-CPI+, rPIV5VΔC, rPIV-Rev, rPIV5-RL, rPIV5-P-S157A,rPIV5-P-S308A, rPIV5-L-A1981D and rPIV5-F-S443P, rPIV5-MDA7,rPIV5ΔSH-CPI−, or rPIV5ΔSH-Rev, and combinations thereof.

In some embodiments of the methods described herein, the PIV5 furtherincludes nucleotide sequences encoding a tumor killing heterologouspolypeptide and/or heterologous RNA. In some embodiments, theheterologous polypeptide is MDA7.

The present invention includes an oncolytic agent including arecombinant or mutant parainfluenza virus 5 (PIV5).

In some embodiments, the oncolytic agent includes one or more themutations selected a mutation of the V/P gene, a mutation of the sharedN-terminus of the V and P proteins, a mutation of residues 26, 32, 33,50, 102, and/or 157 of the shared N-terminus of the V and P proteins, amutation lacking the C-terminus of the V protein, a mutation lacking thesmall hydrophobic (SH) protein, a mutation of the fusion (F) protein, amutation of the phosphoprotein (P), a mutation of the large RNApolymerase (L) protein, a mutation incorporating residues from canineparainfluenza virus, and/or a mutation that enhances synctial formation.

In some embodiments, a mutation includes rPIV5-V/P-CPI−, rPIV5-CPI−,rPIV5-CPI+, rPIV5VΔC, rPIV-Rev, rPIV5-RL, rPIV5-P-S157A, rPIV5-P-S308A,rPIV5-L-A1981D and rPIV5-F-S443P, rPIV5-MDA7, rPIV5ΔSH-CPI−, rPIV5ΔSH-Rev, or combinations thereof.

In some embodiments, the PIV5 further includes nucleotide sequencesencoding a tumor killing polypeptide or RNA. In some embodiments, theheterologous polypeptide is MDA7.

In some embodiments, the oncolytic agent further expresses a fluorescentpolypeptide or detectable agent.

The present invention includes compositions including an oncolytic agentas described herein and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show infection of MDA-MB-435 by PIV5. FIG. 1A showsinfection of MDA-MB-435 cells with PIV5. MDA-MB-435 breast cancer cellsin culture were infected with PIV5 at a multiplicity of infection (MOI)of 10. Mock infection consisted of DMED with 1% BSA. The cells werephotographed at 2 and 3 days post infection (dpi). FIG. 1B shows growthof PIV5 in the cells. Aliquots of cell culture media were also collectedat 0, 48 and 72 hours post infection. Titers of virus in the media weredetermined using plaque assay.

FIGS. 2A and 2B show efficacy of PIV5 as an oncolytic agent in a breastcancer model. FIG. 2A shows efficacy of PIV5. Breast cancer cellsMDA-MB-435 (5×10⁶ cells) were injected into 4 to 6-week old nude micesubcutaneously in the right flank. When the volume (length×width²/2) oftumors reached 125 to 175 mm³ on average, PBS or PIV5 at 1×10⁶ plaqueforming unit (pfu) viruses were directly injected into the tumors once aweek for 5 weeks. The tumor volume was monitored. Shown are the averagetumor volumes +/−SD; N=8 for each group. p≤0.005 for all viruses versusPBS. In FIG. 2B, the experiment was carried out as in FIG. 2A. When thevolume (length×width²/2) of tumors reach 125-175 mm³ on average, PBS orPIV5 at 5×10⁶ (High), 5×10⁵ (Med), and 5×10⁴ (Low) plaque forming unit(pfu) were directly injected into tumors once at one week intervals, for5 weeks. The tumor volume was monitored. N=8 for each group. p≤0.005 forall viruses vs. PBS.

FIGS. 3A and 3B show toxicity of PIV5 in nude mice. Nude mice wereinjected through the tail vein with PBS, or PIV5 (10⁶ pfu) (N=12 foreach group). In FIG. 3A, the weights were measured at 3, 6, 10, 14 and21 days post injection. The average weight of mice on the day ofinjection was set as 100 percent. Plotted are the averages of each group+/−S.D. FIG. 3B shows H & E staining of lungs from infected animals at 6days post infection. Minimal to no inflammation was observed in thesections.

FIGS. 4A-4C show replication of PIV5 in tumors in vivo. FIG. 4A showsdetection of viral RNA in tumors in vivo. The RT-PCR reactions usingprimers specific for PIV5 genome or mRNA were carried out with tumorscollected from the animals. Neg, negative control for RT-PCR; Pos,positive control for RT-PCR. FIG. 4B shows detection of PIV5 in tumorsin vivo using a plaque assay. Breast cancer tumors were grown in nudemice as described in the methods section. The tumors were injected withPIV5 (10⁶ pfu), collected 1, 2, and 6 days post injection, homogenizedand directly applied in a plaque assay to determine the titers ofviruses. The titer of virus from each individual tumor is graphed. InFIG. 4C, mice with MDA-MB-435 tumors were injected with rPIV5-RLat 6×10⁵pfu directly in the tumor. 50 μl of the substrate coelenterazine at 400μg/ml was injected in the tumor at different times after virusinjection. All images recorded at same settings: Bin M (4), f1, 2 m, min5e+04, Max 2e+06. Mouse A is control, which was injected with PBS. MouseB and C were injected with virus. Tumors in mouse B after virusinjection at different times were enlarged.

FIGS. 5A and 5B show spread of virus from tumors in vivo. In FIG. 5A,mice with MDA-MB-435 cells inoculated in two locations (about 15 mm awayfrom each other) developed tumors. The tumor closest to the forelimb wasinjected with virus. 50 μl of substrate coelenterazine was injected inboth tumors on different days and images were recorded. A tumor whichdeveloped from MDA-MB-435 (dashed circle, top) was injected withrPIV5-RL (×10⁵ pfu). In FIG. 5B, at 1 day post injection, 1×10⁶MDA-MB-435 cells were injected at about 10 mm away from the tumor in 100μl volume (dashed circle, bottom). About 4 hours (hrs) later 50 μl ofcoelenterazine at 400 μg/ml was injected in the tumor at the newinoculation site. Substrate injections were repeated at 3 days postvirus injection. These images were recorded using an IVIS camera withthe same settings: Bin M (4), f1, 2 m, color bar: min 5e+04, Max 2e+06.Mouse A represents control with PBS injection, mouse B and C representrPIV5-RL injection.

FIG. 6 shows replication of PIV5 in normal cells vs. cancer cells invivo. Nude mice were injected with hTERT (top circle) or MDA-MB-435(bottom circle) cells. The cells were then injected with PBS (leftmouse) or rPIV5-RL (middle and right mouse). At 1 day post injection,luciferase expression was measured using an IVIS camera.

FIGS. 7A-7D show the effect of PIV5 on tumors in vivo. Photomicrographsof H&E stained sections of four subcutaneously implanted tumors (cellline MDA-MB-435) illustrating areas of necrosis. FIGS. 7A and 7B are PBSinjected tumors (control) at a magnification of 200× and 400×respectively. FIGS. 7C and 7D are PIV5 injected tumors with arrowsindicating syncytia formation at a magnification of 200× and 400×respectively.

FIG. 8 shows the efficacy of a mutant PIV5 as an oncolytic agent. Breastcancer cells MDA-MB-435 (5×10⁶ cells) were injected into 6 to 8-week oldnude mice subcutaneously. PBS, PIV5 or rPIV5-CPI− at 1×10⁶plaque formingunit (pfu) viruses were directly injected into tumors once at one weekinterval. The tumor volume was monitored. Shown are the average tumorvolumes=/−SD. N=8 for each group. p≤0.005 for all viruses vs. PBS.

FIG. 9 shows the efficacy of PIV5 as an oncolytic agent in a melanomamodel. Melanoma cancer cells UACC 903 (10⁶ cells) were injected into 6to 8-week old nude mice subcutaneously. When the volume(length×width²/2) of tumors reached about 100 mm³ on average (about 2weeks), PBS or PIV5 at 1×10⁶ pfu were directly injected into tumors onceat one week interval. The tumor volume was monitored. n=8 for eachgroup. p≤0.005. Shown are the average tumor volumes=/−SD.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Parainfluenza virus 5 (PIV5), a negative-stranded RNA virus, is a memberof the Rubulavirus genus of the family Paramyxoviridae which includesmany important human and animal pathogens such as mumps virus, humanparainfluenza virus type 2 and type 4, Newcastle disease virus, Sendaivirus, HPIV3, measles virus, canine distemper virus, rinderpest virusand respiratory syncytial virus. PIV5 was previously known as simianvirus-5 (SV5) (Chatziandreou et al., 2004, J Gen Virol; 85:3007-3016).Although PIV5 was originally isolated from cultured primary monkey cellsits natural host is the dog in which it causes kennel cough (McCandlishet al., 1978, Vet Rec; 102:293-301). Although PIV5 can infect humans(Cohn et al., 1996, Pathobiology; 64:131-135), no known symptoms ordiseases in humans have been associated with PIV5. Unlike mostparamyxoviruses, PIV5 can infect normal cells with little cytopathiceffect.

With the present invention, it has been discovered that wild type PIV5and mutants thereof can function as oncolytic agents, killing a varietyof tumor cells, including, but not limited to, breast cancer andmelanoma tumor cells. In some aspects, a PIV5 oncolytic agentdemonstrates an oncolytic effect only tumor cells and not effectingnormal cells. A PIV5 oncolytic agent may demonstrate oncolytic activityagainst any of a variety of vertebrate cells. Oncolytic activityincludes, for example, tumor cell death, reduction of tumor cell growth,reduction in tumor size, the induction of tumor cells syncytiaformation, and/or the induction apoptosis in tumor cells. Oncolyticactivity may be assayed by any of a variety of known methods, including,but not limited to any of those described in the examples sectionincluded herewith.

An oncolytic agent of the present invention includes any of a variety ofwild type PIV5 strains, mutant PIV5, or recombinant PIV5 (rPIV5). Wildtype strains include, but are not limited to, the PIV5 strains W3A, WR(ATCC® Number VR-288™), canine parainfluenza virus strain 78-238 (ATCCnumber VR-1573) (Evermann et al., 1980, J Am Vet Med Assoc;177:1132-1134; and Evermann et al., 1981, Arch Virol; 68:165-172),canine parainfluenza virus strain D008 (ATCC number VR-399) (Binn etal., 1967, Proc Soc Exp Biol Med; 126:140-145), MIL, DEN, LN, MEL,cryptovirus, CPI+, CPI−, H221, 78524, T1 and SER. See, for example,Chatziandreou et al., 2004, J Gen Virol; 85(Pt 10):3007-16; Choppin,1964, Virology: 23:224-233; and Baumgartner et al., 1987, Intervirology;27:218-223. Additionally, PIV5 strains used in commercial kennel coughvaccines, such as, for example, BI, FD, Merck, and Merial vaccines, maybe used.

A PIV5 oncolytic agent may be constructed using any of a variety ofmethods, including, but not limited to, the reverse genetics systemdescribed in more detail in He et al. (Virology; 237(2):249-60, 1997).

A PIV5 oncolytic agent may include one, two, three, four, five, or moremutations, including, but not limited to any of those described herein.In some aspects, a combination of two or more (two, three, four, five,six, seven, or more) mutations may be advantageous and may demonstratedenhanced oncolytic activity.

FIG. 1 shows the PIV5 genome structure. PIV5 encodes eight viralproteins. Nucleocapsid protein (NP), phosphoprotein (P) and large RNApolymerase (L) protein are important for transcription and replicationof the viral RNA genome. The V protein plays important roles in viralpathogenesis as well as viral RNA synthesis. The fusion (F) protein, aglycoprotein, mediates both cell-to-cell and virus-to-cell fusion in apH-independent manner that is essential for virus entry into cells. Thestructures of the F protein have been determined and critical amino acidresidues for efficient fusion have been identified. Thehemagglutinin-neuraminidase (HN), another viral glycoprotein, is alsoinvolved in virus entry and release from the host cells. The matrix (M)protein plays an important role in virus assembly and budding. Thehydrophobic (SH) protein is a 44-residue hydrophobic integral membraneprotein and is oriented in membranes with its N terminus in thecytoplasm. For reviews of the molecular biology of paramyxoviruses see,for example, Whelan et al., 2004, Curr Top Microbiol Immunol;283:61-119; and Lamb & Parks, (2006). Paramyxoviridae: the viruses andtheir replication. In Fields Virology, 5th edn, pp. 1449-1496. Edited byD. M. Knipe & P. M. Howley. Philadelphia, Pa.: Lippincott Williams &Wilkins. An oncolytic agent may have a mutation in one or more of theseeight proteins.

PIV5 can infect human (Hsiung et al., 1965, J Immunol; 94:67-73), but ithas not been associated with any known illness. PIV5 infects mice andhamsters but does not cause any symptoms in the animals. PIV5 can begrown in cells and released to media at a titer up to 8×10⁸ pfu/ml,indicating its potential as a safe gene delivery vector and a possiblecost effective way for mass production of the virus.

PIV5 can infect cells productively with little cytopathic effect (CPE)in many cell types. In some cell types, PIV5 infection causes formationof syncytia, i.e., fusion of many cells together, leading to cell death.A mutation may include one or more mutations that promote syncytiaformation (see, for example Paterson et al., 2000, Virology; 270:17-30).

PIV5 infection does not induce apoptosis (He et al., 2001, J Virol;75:4068-4079. However, recombinant PIV5 lacking SH (rPIV5ΔASH) inducesapoptosis in L929 cells through a tumor necrosis factor (TNF)-α mediatedextrinsic apoptotic pathway (He et al., 2001, J Virol; 75:4068-4079; Heet al., 1998, Virology; 250:30-40; and Lin et al., 2003, J Virol;77:3371-3383).

The V protein of PIV5 plays a critical role in blocking apoptosisinduced by virus. Recombinant PIV5 lacking the conserved cysteine-richC-terminus (rPIV5VΔC) of the V protein induces apoptosis in a variety ofcells through an intrinsic apoptotic pathway, likely initiated throughendoplasmic reticulum (ER)-stress (Sun et al., 2004, J Virol;78:5068-5078). Mutant recombinant PIV5 with mutations in the N-terminusof the V/P gene products, such as rPIV5-CPI−, also induce apoptosis(Wansley and Parks, 2002, J Virol; 76:10109-10121). A mutation includes,but is not limited to, rPIV5ΔSH, rPIV5-CPI−, rPIV5VΔC, and combinationsthereof.

A mutation includes, but is not limited to, a mutation of the V/P gene,a mutation of the shared N-terminus of the V and P proteins, a mutationof residues 26, 32, 33, 50, 102, and/or 157 of the shared N-terminus ofthe V and P proteins, a mutation lacking the C-terminus of the Vprotein, a mutation lacking the small hydrophobic (SH) protein, amutation of the fusion (F) protein, a mutation of the phosphoprotein(P), a mutation of the large RNA polymerase (L) protein, a mutationincorporating residues from canine parainfluenza virus, and/or amutation that enhances synctial formation.

A mutation may include, but is not limited to, rPIV5-V/P-CPI−,rPIV5-CPI−, rPIV5-CPI+, rPIV5VΔC, rPIV-Rev, rPIV5-RL, rPIV5-P-S157A,rPIV5-P-S308A, rPIV5-L-A1981D and rPIV5-F-S443P, rPIV5-MDA7,rPIV5ΔSH-CPI−, rPIV5ΔSH-Rev, and combinations thereof.

An oncolytic agent includes a recombinant PIV5 construct including anyone or more of the mutations described herein, including one or more ofthe constructs described the in examples section included herewith.

Also included with the present invention are PIV5 oncolytic agentsincluding heterologous nucleotide sequences. Such a heterologousnucleotide sequence may encode, for example, a heterologous DNA, RNA, orpolypeptide. In some aspects, a PIV5 oncolytic agent includingheterologous nucleotide sequences may also include one or more of themutations described herein.

A heterologous nucleotide sequence may be tumor killing agent, forexample, a tumor suppressor gene. A tumor suppressor gene is a gene thatregulates the growth of cells and, thus, can prevent and inhibit thegrowth of tumors. A tumor suppressor gene may, for example, slow or stopcell division, repair DNA damage, or regulate cell death via apoptosis.Examples include, but are not limited to, melanomadifferentiation-associated gene-7 (MDA-7), p53, BRCA1 (BReast Cancer-1)and BRCA2, ATM (ataxia telangiectasia), retinoblastomia tumor suppressorgene (RB), and TSG101. The melanoma differentiation-associated gene-7(MDA-7) protein, also known as interleukin (IL)-24, is a tumorsuppressor that induces apoptosis in a variety of human malignant cellsincluding lung cancer cells (Ishikawa et al., 2005, Clin Cancer Res;11:1198-1202).

In some aspects, an oncolytic agent may express a fluorescentpolypeptide or detectable agent. Such an agent may be used in diagnosticagents, for examples methods of imaging tumors, including for example,primary tumors and/or metastatic tumors.

Also included in the present invention are compositions including one ormore of the viral constructs, as described herein. Such a compositionmay include a pharmaceutically acceptable carrier. As used, apharmaceutically acceptable carrier refers to one or more compatiblesolid or liquid fillers, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Sucha carrier may be pyrogen free. The present invention also includesmethods of making and using the oncolytic agents and compositionsdescribed herein.

The compositions of the present disclosure may be formulated inpharmaceutical preparations in a variety of forms adapted to the chosenroute of administration. One of skill will understand that thecomposition will vary depending on mode of administration and dosageunit. The agents of this invention can be administered in a variety ofways, including, but not limited to, intravenous, topical, oral,subcutaneous, intraperitoneal, intramuscular, and intratumor deliver. Insome aspects, the agents of the present invention may be formulated forcontrolled or sustained release. In some aspects, a formulation forcontrolled or sustained release is suitable for subcutaneousimplantation. In some aspects, a formulation for controlled or sustainedrelease includes a patch. An agent may be formulated for enteraladministration, for example, formulated as a capsule or tablet.

An oncolytic agent of the present disclosure may be administered to apatient in methods for the treatment of cancer. Cancers to be treatedinclude, but are not limited to, melanoma, basal cell carcinoma,colorectal cancer, pancreatic cancer, breast cancer, prostate cancer,lung cancer (including small-cell lung carcinoma and non-small-cell lungcarcinoma), leukemia, lymphoma, sarcoma, ovarian cancer, Kaposi'ssarcoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, multiple myeloma,neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primarymacroglobulinemia, small-cell lung tumors, primary brain tumors, stomachcancer, head and neck cancers, malignant pancreatic insulanoma,malignant carcinoid, urinary bladder cancer, premalignant skin lesions,testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophagealcancer, genitourinary tract cancer, malignant hypercalcemia, cervicalcancer, kidney cancer, endometrial cancer, glioblastoma, mesothelioma,oral leukoplakia, Barrett's esophageal cancer, and adrenal corticalcancer. In some aspects, the cancer is a primary cancer. In someaspects, the cancer is metastatic, including, but not limited to ametastatic melanoma, metastatic breast cancer, or metastatic colorectalcancer.

The PIV5 viral constructs described herein are useful as oncolyticagents. The present invention provides methods for killing tumor cells,reducing the growth of tumor cells, reducing tumor size, inducingapoptosis in a tumor cell, and/or inducing tumor cells syncytialformation. The present invention also provides methods for killing tumorcells, reducing the growth of tumor cells, reducing tumor size, inducingapoptosis in a tumor cell, inducing tumor cells syncytia formation,inhibiting tumorigenesis, and/or treating cancer in a subject. Thisinvolves administering to a patient an effective amount of an oncolyticagent or composition, as described herein.

The efficacy of such methods for the treatment of cancer may be assessedby any of various parameters well known in the art. This includes, butis not limited to, determinations of a reduction in tumor size,determinations of the inhibition of the growth, spread, invasiveness,vascularization, angiogenesis, and/or metastasis of a tumor,determinations of the inhibition of the growth, spread, invasivenessand/or vascularization of any metastatic lesions, determinations oftumor infiltrations by immune system cells, and/or determinations of anincreased delayed type hypersensitivity reaction to tumor antigen. Theefficacy of treatment may also be assessed by the determination of adelay in relapse or a delay in tumor progression in the subject or by adetermination of survival rate of the subject, for example, an increasedsurvival rate at one or five years post treatment. As used herein, arelapse is the return of a tumor or neoplasm after its apparentcessation.

Several other viruses have been successfully used as oncolytic agentsfor treating various cancers in animal model systems and some of themare in clinical trials. For example, adenovirus, measles virus (MeV) andNewcastle disease virus (NDV) have are currently being tested inclinical trials. However, the PIV5-based oncolytic agents of the presentinvention present advantages. While PIV5 is a virus that infects manyanimals and humans, is not associated with any human diseases. PIV5causes syncytia formation in tumor cells, leading to cancer cell death.And, as a negative stranded RNA virus, PIV5 is unable to integrate intothe host genome.

As used herein “treating” or “treatment” can include therapeutic and/orprophylactic treatments. “Treating a disorder,” as used herein, is notintended to be an absolute term. Treatment may lead to an improvedprognosis or a reduction in the frequency or severity of symptoms.Desirable effects of treatment include preventing occurrence orrecurrence of disease, alleviation of symptoms, and/or diminishment ofany direct or indirect pathological consequences of the disease,decreasing the rate of disease progression, amelioration or palliationof the disease state, and remission or improved prognosis. Likewise, theterm “preventing,” as used herein, is not intended as an absolute term.Instead, prevention refers to delay of onset, reduced frequency ofsymptoms, or reduced severity of symptoms associated with a disorder.Prevention therefore refers to a broad range of prophylactic measuresthat will be understood by those in the art. In some circumstances, thefrequency and severity of symptoms is reduced to non-pathologicallevels. In some circumstances, the symptoms of an individual receivingthe compositions of the invention are only 90, 80, 70, 60, 50, 40, 30,20, 10, 5 or 1% as frequent or severe as symptoms experienced by anuntreated individual with the disorder.

An oncolytic agent may be administered as a composition. Compositionsmay be administered in any of the methods of the present invention andmay be formulated in a variety of forms adapted to the chosen route ofadministration. The formulations may be conveniently presented in unitdosage form and may be prepared by methods well known in the art ofpharmacy. A composition may include a pharmaceutically acceptablecarrier. The term “pharmaceutically acceptable,” as used herein, meansthat the compositions or components thereof so described are suitablefor use in contact with human skin without undue toxicity,incompatibility, instability, allergic response, and the like. Acomposition may be a pharmaceutical composition.

Oncolytic agents, as described herein, can be administered by anysuitable means including, but not limited to, for example, parenteral(involving piercing the skin or mucous membrane), oral (through thedigestive tract), transmucosal, rectal, nasal, topical (including, forexample, transdermal, aerosol, buccal and sublingual), or vaginal.Administration may include, for example, subcutaneous, intramuscular,intravenous, intradermal, intraperitoneal, intrasternal, intraarticularinjections, intravesical, intra-arteriole, intraventricular,intracranial, intranasal, oral, in situ, intratumoral, by inhalation, orintralesional (for example, by injection into or around a tumor) as wellas various infusion techniques.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intraperitoneal, and intratumoraladministration. In this connection, sterile aqueous media that can beemployed will be known to those of skill in the art. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.

For human and veterinary administration, oncolytic agents, as describedherein, may meet sterility, pyrogenicity, and general safety and puritystandards as required by the FDA. Such compositions are consideredsuitable for parenteral or enteral administration to a mammal. Suchcompositions may be pyrogen-free.

For enteral administration, an agent may be administered in a tablet orcapsule, which may be enteric coated, or in a formulation for controlledor sustained release. Many suitable formulations are known, includingpolymeric or protein microparticles encapsulating drug to be released,ointments, gels, or solutions which can be used topically or locally toadminister drug, and even patches, which provide controlled release overa prolonged period of time. These can also take the form of implants.Such an implant may be implanted within the tumor.

Therapeutically effective concentrations and amounts may be determinedfor each application herein empirically by testing the compounds inknown in vitro and in vivo systems, such as those described herein,dosages for humans or other animals may then be extrapolated therefrom.

An agent of the present disclosure may be administered at once, or maybe divided into a number of multiple doses to be administered atintervals of time. For example, agents of the invention may beadministered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or moretimes, or may be administered by continuous infusion. It is understoodthat the precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by extrapolation from in vivo or in vitro testdata. It is to be noted that concentrations and dosage values may alsovary with the severity of the condition to be alleviated. It is to befurther understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that any concentrationranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed compositions and methods.

By a “therapeutically effective amount” is meant a sufficient amount ofthe compound to treat the subject at a reasonable benefit/risk ratioapplicable to obtain a desired therapeutic response. It will beunderstood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including, for example, the disorderbeing treated and the severity of the disorder, activity of the specificcompound employed, the specific composition employed, the age, bodyweight, general health, sex and diet of the patient, the time ofadministration, route of administration, and rate of excretion of thespecific compound employed, the duration of the treatment, drugs used incombination or coincidentally with the specific compound employed, andlike factors well known in the medical arts.

In some therapeutic embodiments, an “effective amount” of an agent is anamount that results in a reduction of at least one pathologicalparameter. Thus, for example, in some aspects of the present disclosure,an effective amount is an amount that is effective to achieve areduction of at least about 10%, at least about 15%, at least about 20%,or at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,or at least about 95%, compared to the expected reduction in theparameter in an individual not treated with the agent.

An oncolytic agent as described herein may be used in methods of imagingtumor cells. An oncolytic agent as described herein may be used inmethods of imaging a tumor in a subject. For such applications, anoncolytic agent may express a fluorescent polypeptide or otherdetectable agent.

An oncolytic agent as described herein may be used for the expression ofa heterologous nucleotide sequence or polypeptide sequence within a cellor tumor.

As used herein, the term “subject” represents an organism, including,for example, a mammal. A mammal includes, but is not limited to, ahuman, a non-human primate, and other non-human vertebrates. A subjectmay be an “individual,” “patient,” or “host.” Non-human vertebratesinclude livestock animals (such as, but not limited to, a cow, a horse,a goat, and a pig), a domestic pet or companion animal, such as, but notlimited to, a dog or a cat, and laboratory animals. Non-human subjectsalso include non-human primates as well as rodents, such as, but notlimited to, a rat or a mouse. Non-human subjects also include, withoutlimitation, poultry, horses, cows, pigs, goats, dogs, cats, guinea pigs,hamsters, mink, and rabbits.

As used herein “in vitro” is in cell culture and “in vivo” is within thebody of a subject. As used herein, “isolated” refers to material thathas been either removed from its natural environment (e.g., the naturalenvironment if it is naturally occurring), produced using recombinanttechniques, or chemically or enzymatically synthesized, and thus isaltered “by the hand of man” from its natural state.

In some aspects of the methods of the present invention, a methodfurther includes the administration of one or more additionaltherapeutic agents. One or more additional therapeutic agents may beadministered before, after, and/or coincident to the administration ofan oncolytic agent described herein. An oncolytic agent as describedherein and additional therapeutic agents may be administered separatelyor as part of a mixture or cocktail. In some aspects of the presentinvention, the administration of an oncolytic agent may allow for theeffectiveness of a lower dosage of other therapeutic modalities whencompared to the administration of the other therapeutic modalitiesalone, providing relief from the toxicity observed with theadministration of higher doses of the other modalities.

As used herein, an additional therapeutic agent may be an agent whoseuse for the treatment of cancer is known to the skilled artisan.Additional therapeutic treatments include, but are not limited to,surgical resection, radiation therapy, hormone therapy, vaccines,antibody based therapies, whole body irradiation, bone marrowtransplantation, peripheral blood stem cell transplantation, theadministration of chemotherapeutic agents (also referred to herein as“antineoplastic chemotherapy agent,” “antineoplastic agents,” or“antineoplastic chemotherapeutic agents”), cytokines, antiviral agents,immune enhancers, tyrosine kinase inhibitors, protein kinase C (PKC)modulator (such as, for example, the PKC activator ingenol 3-angelate(PEP005) or the PKC inhibitor bisindolylmaleimid (enzastaurin), signaltransduction inhibitors, antibiotics, antimicrobial agents, a TLRagonist (such as for example, bacterial lipopolysaccharides (LPS) or aCpG oligonucleotide (ODN)), an inhibitor of IDO, such as, for example,1-MT, and adjuvants.

A chemotherapeutic agent may be, for example, a cytotoxic chemotherapyagent, such as, for example, epidophyllotoxin, procarbazine,mitoxantrone, platinum coordination complexes such as cisplatin andcarboplatin, leucovorin, tegafur, paclitaxel, docetaxol, vincristine,vinblastine, methotrexate, cyclophosphamide, gemcitabine, estramustine,carmustine, adriamycin (doxorubicin), etoposide, arsenic trioxide,irinotecan, epothilone derivatives, navelbene, CPT-11, anastrazole,letrazole, capecitabine, reloxafine, ifosamide, and droloxafine.

A chemotherapeutic agent may be, for example, an alkylating agent, suchas, for example, irofulven, nitrogen mustards (such as chlorambucil,cyclophosphamide, ifosfamide, mechlorethamine, melphalan, and uracilmustard), aziridines (such as thiotepa), methanesulphonate esters (suchas busulfan), nitroso ureas (such as carmustine, lomustine, andstreptozocin), platinum complexes (such as cisplatin and carboplatin),and bioreductive alkylators (such as mitomycin, procarbazine,dacarbazine and altretamine), ethylenimine derivatives, alkylsulfonates, triazenes, pipobroman, temozolomide, triethylene-melamine,and triethylenethiophosphoramine.

A chemotherapeutic agent may be an antimetabolite, such as, for example,a folate antagonist (such as methotrexate and trimetrexate), apyrimidine antagonist (such as fluorouracil, fluorodeoxyuridine, CB3717,azacitidine, cytarabine, gemcitabine, and floxuridine), a purineantagonist (such as mercaptopurine, 6-thioguanine, fludarabine, andpentostatin), a ribonucleotide reductase inhibitor (such ashydroxyurea), and an adenosine deaminase inhibitor.

A chemotherapeutic agent may be a DNA strand-breakage agent (such as,for example, bleomycin), a topoisomerase II inhibitor (such as, forexample, amsacrine, dactinomycin, daunorubicin, idarubicin,mitoxantrone, doxorubicin, etoposide, and teniposide), a DNA minorgroove binding agent (such as, for example, plicamydin), a tubulininteractive agent (such as, for example, vincristine, vinblastine, andpaclitaxel), a hormonal agent (such as, for example, estrogens,conjugated estrogens, ethinyl estradiol, diethylstilbesterol,chlortrianisen, idenestrol, progestins (such as hydroxyprogesteronecaproate, medroxyprogesterone, and megestrol), and androgens (such astestosterone, testosterone propionate, fluoxymesterone, andmethyltestosterone)), an adrenal corticosteroid (such as, for example,prednisone, dexamethasone, methylprednisolone, and prednisolone), aleutinizing hormone releasing agent or gonadotropin-releasing hormoneantagonist (such as, for example, leuprolide acetate and goserelinacetate), an antihormonal agent (such as, for example, tamoxifen), anantiandrogen agent (such as flutamide), an antiadrenal agent (such asmitotane and aminoglutethimide), and a natural product or derivativethereof (such as, for example, vinca alkaloids, antibiotics, enzymes andepipodophyllotoxins, including, for example vinblastine, vincristine,vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin,epirubicin, idarubicin, ara-C, paclitaxel, mithramycin,deoxyco-formycin, mitomycin-C, L-asparaginase, and teniposide.

In some aspects of the methods of the present invention, at least oneadditional therapeutic agent includes radiation therapy. In someaspects, radiation therapy includes localized radiation therapydelivered to the tumor. In some aspects, radiation therapy includestotal body irradiation.

Cytokines include, but are not limited to, IL-1α, IL-1β, IL-2, IL-3,IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-19, IL-20,IFN-α, IFN-β, IFN-γ, tumor necrosis factor (TNF), transforming growthfactor-β (TGF-β), granulocyte colony stimulating factor (G-CSF),macrophage colony stimulating factor (M-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), and or Flt-3 ligand. Antibodytherapeutics, include, for example, trastuzumab (Herceptin) andantibodies to cytokines, such as IL-10 and TGF-β.

In some aspects of the methods of the present invention, the oncolyticagent administered may include any one or more of the PIV5-basedconstructs described in International Application No. PCT/US2013/022962,titled “Parainfluenza Virus 5 Based Vaccines,” inventor Biao He, filedJan. 24, 2013, which is hereby incorporated by reference herein in itsentirety.

In some aspects of the methods of the present invention, a measurementof response to treatment observed after administering both an oncolyticagent as described herein and the additional therapeutic agent isimproved over the same measurement of response to treatment observedafter administering either the oncolytic agent or the additionaltherapeutic agent alone. In some aspects of the methods of the presentinvention, the administration an oncolytic as described herein and theat least one additional therapeutic agent demonstrate therapeuticsynergy. As used herein, a combination may demonstrate therapeuticsynergy if it is therapeutically superior to one or other of theconstituents used at its optimum dose (Corbett et al., 1982, CancerTreatment Reports; 66:1187. In some embodiments, a combinationdemonstrates therapeutic synergy if the efficacy of a combination ischaracterized as more than additive actions of each constituent.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Parainfluenza Virus 5, an Oncolytic Reagent forBreast Cancer

This example demonstrates the ability of PIV5 to prevent the growth ofhuman breast cancer cells in vitro and in vivo in a xenograft model.Live imaging, viral titers and RT-PCR were used to follow the fate ofthe virus in vivo. PIV5 inhibited the growth of MDA-MB-435 cells inculture and tumors in nude mice. It appeared to do so by causingsyncytia formation of tumor cell. When PIV5 was inoculated into asubcutaneously growing tumor, the virus replicated and spread tonon-adjacent tumors. The virus was selective; i.e. it did not infectnormal, surrounding cells. Furthermore, tail vein injection of high doseof PIV5 did not cause weight loss or illness. Because the PIV5 genomecan be modified, it is possible to further enhance its oncolyticactivity by changing its genome and by inserting tumor-killing proteinsor RNA. Thus, PIV5 is to be considered as a potential oncolytic agent.

Viruses in the Paramyxoviridae family of Mononegavirales are negativestranded, non-segmented RNA viruses (NNSV), including mumps virus (MuV),Newcastle disease virus (NDV) and measles virus (MeV), which are knownto be effective in reducing tumors in humans (see, for example,Fielding, 2005, Rev Med Virol; 15:135-142; and Myers et al., 2005,Cancer Gene Ther; 12:593-599). PIV5, formerly known as simian virus 5(SV5) (Chatziandreou et al., 2004, J Gen Virol; 85:3007-3016), is amember of the Rubulavirus genus of the family Paramyxoviridae. AlthoughPIV5 was originally isolated from cultured primary monkey cells itsnatural host is thought to be the dog in which it causes kennel cough(McCandlish et al., 1978, Vet Rec; 102:293-301). PIV5 can infect humans(Cohn et al., 1996, Pathobiology; 64:131-135), but with no knownsymptoms or diseases (Hsiung et al., 1965, J Immunol; 94:67-73).

PIV5 has seven genes but encodes eight known viral proteins.Nucleocapsid protein (NP), phosphoprotein (P) and large RNA polymerase(L) protein are important for transcription and replication of the viralRNA genome. The V/P gene of PIV5 is transcribed into both the V mRNA andthe P mRNA through the process of “RNA editing.” The V mRNA is made whenthe viral RNA polymerase faithfully transcribes the V/P gene. However,during transcription the viral RNA polymerase complex recognizes aspecific RNA sequence in the V/P gene and inserts two non-templated Gresidues at the site to generate the P mRNA (Thomas et al., 1988, Cell;54:891-902). As a result, the V/P gene is transcribed into two mRNAs atabout the same level and translated into two proteins, which shareidentical N-termini but different C-termini. The V protein playsimportant roles in viral pathogenesis as well as viral RNA synthesis(Didcock et al., 1999, J Virol; 73:9928-9933; Andrejeva et al., 2002, JVirol; 76:2159-2167; Poole et al. 2002, Virology; 303:33-46; He et al.,2002, Virology; 303:15-32; Precious et al., 2005, J Virol;79:13434-13441; Lin and Lamb, 2000, J Virol; 74:9152-9166; Lin et al.,2005, Virology; 338:270-280; and Lin et al., 2007, Virology;368:262-272).

The fusion (F) protein, a glycoprotein, mediates both cell-to-cell andvirus-to-cell fusion in a pH-independent manner that is essential forvirus entry into cells. The structures of the F protein have beendetermined and critical amino acid residues for efficient fusion havebeen identified (Yin et al., 2006, Nature; 439:38-44; and Paterson etal., 2000, Virology; 270:17-30). The hemagglutinin-neuraminidase (HN),another glycoprotein, is also involved in virus entry and release fromthe host cells. The matrix (M) protein is important for virus assemblyand budding (Schmitt et al. 1999, J Virol; 73:8703-8712; and Schmitt etal., 2002, J Virol; 76:3952-3964). The small hydrophobic (SH) protein isan integral membrane protein oriented with its N terminus in thecytoplasm (Hiebert et al., 1988, J Virol; 62:2347-2357). Recent workindicates that SH plays a role in blocking TNF-α-mediated apoptosis (Linet al., 2003, J Virol; 77:3371-3383; and Fuentes et al., 2007, J Virol;81:8361-8366).

This example demonstrated that PIV5 was effective as an anti-tumor agentagainst breast cancer in xenograft model systems and demonstratedsimilar activity in a melanoma model.

MATERIALS AND METHODS

Cells and viruses. MDA-MB-435 breast cancer cells (ATCC) and UACC 903melanoma cancer cells (Trent et al., 1990, Science; 247:568-571 andSharma et al., 2006, Cancer Res; 66:8200-8209) were grown in DMEM mediumcontaining 10% FCS. hTERT (human mammary epithelial cells that areimmortalized with telomerase (HMEC-hTERT, referred as hTERT) (formerlyClontics, CA, now under Lonza, Basel) cells were grown in MGEMserum-free medium (Lonza). Viruses rPIV5 WT, rPIV5-R-Luc, and rPIV5-CPI−mutant viruses were grown in MDBK cells as described previously (He etal., 1997, Virology; 237:249-260). Viruses were titrated in BHK cellsusing a plaque assay. For in vitro infection, MDA-MB-435 cells wereinfected at multiplicity of infection (MOI) of 10 in DMEM containing 1%of BSA for 1 hr followed by a medium change to DMEM containing 2% FCS.An aliquot of cell media was collected at 0, 24, 48 and 72 hours postinfection (hpi) and the virus titers were determined using a plaqueassay.

Animal experiments. All the animal experiments strictly followed theprotocols approved by the Institutional Animal Care and Use Committee ofThe Pennsylvania State University. 5×10⁶ of MDA-MB-435 cells or 1×10⁶ ofUACC 903 cells in 100 μl PBS were injected subcutaneously into the rightflank of 6-8 week old female nude mice. The tumor length and width weremeasured with a caliper. Tumor volumes were calculated aslength×width²/2 (Dethlefsen et al., 1968, J Natl Cancer Inst;40:389-405). When the average tumor volume reached 125-175 mm³, in 3-5weeks, the mice were grouped by tumors of similar average sizes, andrandomly assigned to PBS or PIV5 WT or mutant. Viruses of 10⁶ plaqueforming unit (pfu) or PBS (50 μl) were injected directly into the tumor.The injections were repeated weekly. The weight of mice and the volumeof tumors were monitored. To determine virus toxicity, WT PIV5 virus(10⁶ pfu) or PBS was injected through tail vein. Weight and signs ofillness were monitored up to 21 days. The mice were sacrificed andlungs, livers, hearts, kidneys, and spleens were collected at 3, 6, 10,14, and 21 days after virus injection. The organs were fixed in 4%paraformaldehyde and processed for histological analysis, as describedbelow.

RNA isolation from tumors and RT-PCR. Total RNA was isolated using RNAEasy Kit (Qiagen) from tumors collected one week after the last virusinjection. For viral RNA amplification, RT-PCR was carried out in onetube of RT-PCR (Gene Choice) following the manufacturer's protocol usingPCR primers BH185 and BH186, which hybridize to viral genome andanti-genome respectively. For mRNA RT-PCR, the RT was carried out usingpoly dT oligo and Superscript II (Invitrogen), and the SS cDNA productwas used as template for PCR.

Titration of virus recovered from tumor. Tumors were injected withPIV5-WT virus (10⁶ pfu in 50 μl volume). At 1, 2, and 6 days postinjection, the tumors were collected and homogenized in DMEM. Thesuspensions were used for plaque assays directly.

Live imaging. To examine the virus amplification and spread in vivo, twotumors were grown 15 mm apart by inoculating MDA-MB-435 cells at twolocations in the right flank of a mouse. rPIV5-RLuc of 5×10⁵ pfu in 50μl was injected into the tumor close to the forelimb. The luminescencewas recorded using IVIS (Xenogen, Inc.) live imaging system on 2, 3, and25 days post virus injection. For live imaging, mice were anaesthetizedwith isoflurane, and 400 μg/ml of coelentrazine (made by diluting 1mg/ml stock in methanol with PBS immediately before injection) wasinjected locally. To detect spread of virus in vivo, rPIV5-RLuc (5×10⁵)were injected directly into the flank tumors of MDA-MB-435 cells. At 1day post virus injection, 5×10⁶ MDA-MB-435 cells in 100 μl PBS wereinjected about 10 mm away from the tumor. Four hours later, 50 μl of 400μg/ml of the substrate coelenterazine were injected in the tumor and thecell injection site for live imaging recording. The imaging procedurewas repeated at 3 days post virus injection. For virus infection andreplication in cancer cells and normal cells in vivo, the mice wereinoculated with MDA-MB-435 cells and hTERT cells 10 mm apart on theright flank. The next day, rPIV5-RLuc (5×10⁵) or PBS (as negativecontrol) was injected at both cell sites. One day later, live imaging ofluminescence was recorded.

Histology analysis. Tumor were collected and fixed with 4%paraformaldehyde in PBS at 4° C. Samples were processed in a ShannonCitadel 2000 paraffin processor (Thermo Fisher) following standardprocedures. Processed samples were embedded in paraffin and sectioned at5 μm. The sections were then subjected to H & E staining in a ShandonGemini Varistainer (Thermo Fisher).

Statistical analysis. Statistical analysis was performed using student'st-test. P values of PIV5 vs. PBS in FIG. 2, FIG. 8 and FIG. 9 are lessthan 0.05.

RESULTS

PIV5 infects breast cancer cells. To examine whether PIV5 was a goodcandidate as an anti-tumor agent, the virus was used to infect awell-studied human metastatic breast cancer cell line, MDA-MB-435, invitro. PIV5 readily infected MDA-MB-435 cells (FIG. 1A). At three dayspost infection, the virus grew to a high titer and syncytia wereobserved in the infected cells (FIG. 1B).

Efficacy of PIV5 as an oncolytic agent against breast cancer in axenograft mouse model. The efficacy of the wild type virus in killingMDA-MB-435 breast cancer cells grown subcutaneously in the right flankof nude mice was investigated. When the volume of tumors reached 125 to175 mm³ at about 5-6 weeks, mice were injected with 50 μl PBS or 50 μlwild type PIV5 (10⁶ plaque forming units (pfu)) in situ, weekly. It wasfound that PIV5 was effective in reducing tumor growth starting at 3weeks after the initial injection (FIG. 2A). To further study theeffects of PIV5 on tumor growth, a similar experiment was carried outusing different doses of viruses. The lowest dose (5×10⁴ pfu) was aseffective in reducing tumor growth as the highest dose tested (5×10⁶pfu) (FIG. 2B). Interestingly, the tumors disappeared in 8 out of 25mice injected with PIV5 at the time of experiment termination followingthe humane endpoint protocol (at 7 weeks after initial injection whenthe tumors in the PBS group reached 1,000 mm in diameter).

Toxicity of PIV5 in mice. Previously, it was reported that PIV5infection does not cause disease in immunocompetent mice (Chang andHsiung, 1965, J Immunol; 95:591-601). To evaluate toxicity of PIV5 innude mice, PBS or PIV5 was injected via tail veins, and monitored themice for weight loss and signs of illness. Injections of viruses did notcause weight loss or illness up to 21 days post infection (FIG. 3A).Lung, liver, heart, kidney and spleen of the mice were collected at 3,6, 10, 14 and 21 days after tail vein injections. The organs weresectioned and subjected to H & E staining. A board certified veterinarypathologist found no significant lesions in any of the samples,indicating that PIV5 was safe for immunocompromised mice (FIG. 3B).

Replication of PIV5 in tumors in vivo. To examine whether the virusreplicated in cancer cells in vivo, the tumors were infected with PIV5,collected and assayed for the presence of the viral transcripts andgenome using RT-PCR. Viral transcripts and genomes were detected in thetumors at 6 weeks after the initial injection (FIG. 4A), indicating thatthe virus replicated in the cancer cells in vivo. Because PIV5 is anegative stranded RNA virus, its life cycle does not have a DNA phase,and it is not known to cause latent infection. Hence, detection of viralgenomic (negative sense) RNA and mRNA normally indicates virusreplication. To further prove that the virus replicated in tumors invivo, virus titers were directly measured from extracted tumors. Cancercells were injected into nude mice and about 6 weeks allowed for tumorsto develop. PIV5 was then injected into the tumors, and at 1, 2 and 6days after injection, the tumors were collected and homogenized. Virustiters were measured in the homogenates directly using a plaque assay.We detected virus in tumors at 6 days post infection (dpi) (FIG. 4B).There was an increase in virus titers between 2 dpi and 1 dpi,indicating that the virus had replicated and grown in tumors in vivo.The trend of virus growth continued to 6 dpi, the last point in theexperiment, confirming that the virus replicated in tumors in vivo. Thisresult was consistent with the previous findings of viral genome RNA andviral mRNA in tumors extracted from the mice.

To measure virus replication in tumors in vivo readily and withoutkilling the animals, a recombinant PIV5 was generated that containsrenilla luciferase as an extra gene between the HN and L gene(rPIV5-RL). Tumors were injected with rPIV5-RL and 1 and 3 days later,substrate was injected into the tumors directly and expression levels ofluciferase in tumors in mice were recorded using the IVIS camera.Luciferase activity was observed in rPIV5-RL-injected tumors, confirmingits replication in tumors in vivo in live animals (FIG. 4C).Interestingly, there were increases of the luciferase intensity overtime within the same tumor, suggesting that virus might have spread toother cancer cells within the given tumors. No luciferase activity wasobserved in normal mouse cells, suggesting that rPIV5-RL preferentiallyinfected cancer cells.

Spread of PIV5 in tumors in vivo. In order to target metastastic cells,it is critical that the oncolytic virus can spread in vivo. To furthertest whether PIV5 spread to areas beyond the original injection site,mice with two tumors were used. Virus was injected in one tumor andexamined whether there was expression of luciferase in the other tumor.Indeed, expression of luciferase was observed in the tumor that was notinjected with virus (FIG. 5A), indicating that PIV5 had spread from theoriginal site of injection to other sites. One potential problem withthis experiment was that we were unable to differentiate whether theluciferase activity in the second site was due to virus spread or virusspill during the initial virus injection. It is possible that during theinitial injection, some of the virus entered the bloodstream and spreadto the other tumor through the blood.

To differentiate between these possibilities, a different injectionregimen was used. First virus was injected into tumors as before; waitedfor 24 hours to allow PIV5 that was not inside cells to dissipate; theninjected tumor cells into mice with tumors that had already beeninjected with PIV5. If virus replication was observed in these newlyinjected cancer cells, the virus would likely come from other tumorsthat were injected with virus, indicating that virus spread from tumorcell to tumor cell. Following this new regimen of virus and cellsinjection, expression of luciferase was observed in newly injected cells(FIG. 5B), indicating that PIV5 spread to other sites in vivo.

Both 5 mm and 10 mm distances between tumors injected with virus and thesite of cell injection were used. In both cases, the distant cellsexpressed luciferase. Thus, PIV5 spread at least 10 mm in vivo.Luciferase activity was only observed in tumor cells, and not in normalmouse cells, strongly suggesting that PIV5 preferentially infected tumorcells in vivo.

Replication of PIV5 in normal cells and cancer cells in vivo. Thefinding that no luciferase activity was observed in normal cellsadjacent to tumors (FIG. 4 and FIG. 5) implied that PIV5 preferentiallyinfected cancer cells in vivo. To further test the preferentialinfection of cancer cells over normal cells, normal human mammaryepithelial cells that are immortalized with telomerase (HMEC-hTERT,referred as hTERT) and MDA-MB-435 cells were infected with PIV5 andvirus titers measured. PIV5 grew to 10⁷ to 10⁸ pfu/ml in the cancercells but grew to 10⁵ to 10⁶ pfu/ml in hTERT, indicating that PIV5 grewmuch better in the cancer cells. To compare the growth and replicationof the virus in vivo, nude mice were injected with equal numbers ofhTERT and MDA-MB-435 cells. Three days later, the cells were injectedwith equal numbers of rPIV5-RL infectious particles. Expression ofluciferase was measured one day after infection. rPIV5-RL gave muchhigher levels of luciferase expression in cancer cells than in normalcells, (FIG. 6) indicating that PIV5 preferentially infected cancercells in vivo.

Mechanisms of reduction of PIV5 on tumor growth in vivo. To investigatethe mechanism of reduction of tumor growth in vivo, tumors werecollected after PIV5 or PBS injection. The tumors were fixed, paraffinembedded, sectioned and subjected to H&E staining. While some necrosesof cells in the center of tumors were observed in both PIV5 and PBSinfected tumors, likely due to insufficient supply of nutrients tosustain rapid tumor growth, formation of syncytia were observed only inPIV5 injected cells, suggesting that PIV5 may kill tumor cells bycausing syncytia formation. It appeared that tumor cells were replacedby connective tissue, especially with PIV5 infection (FIG. 7).

Effect of a recombinant PIV5 with mutations in the V/P gene on tumor invivo. A recombinant PIV5 containing six mutations at the sharedN-terminal of V/P proteins of V and P (26, 32, 33, 50 102 and 157)(rPIV5-CPI−) induced cell death and showed accelerated viral geneexpression in vitro (Wansley and Parks, 2002, J Virol; 76:10109-10121).It was tested for its ability to inhibit tumor growth in nude mice. Atabout 6 weeks post-inoculation, when tumors grew to about 125 to 175 mm³in volume, PBS, PIV5 or rPIV5-CPI− were injected into tumors. rPIV5-CPI−reduced tumor growth rate by about 20% compare with PBS (FIG. 8).However, it was not effective as wild type PIV5.

Efficacy of PIV5 against a melanoma model. Because PIV5 was effective inreducing tumor growth in a breast cancer model, whether this oncolyticactivity could be detected with other cancers was investigated. Melanomacancer cells UACC 903 were injected subcutaneously into nude mice andinjected PIV5 into the tumors when their average volume was about 100mm³. PIV5 infection reduced the rate of melanoma growth in vivo,indicating that oncolytic activity of PIV5 extended to melanoma (FIG.9).

DISCUSSION

This example demonstrated that wild type PIV5 was effective in reducingtumor growth in both breast cancer and melanoma model systems, thusproviding a virus as an additional therapeutic agent. Because PIV5 has anon-segmented negative stranded RNA genome, manipulation of the RNAgenome directly is not feasible at the present time. However, a reversegenetics system was developed for PIV5, enabling changes in the PIV5 RNAgenome through manipulation of its cDNA sequence. Using this system,mutations as well as foreign genes have been introduced into the RNAgenome of PIV5 and viable recombinant infectious viruses have beenobtained. Thus, it is conceivable that further modification of PIV5genome will generate more potent oncolytic virus variants. For instance,it has been reported that PIV5 is a good vector for expressing foreigngenes (Tompkins et al., 2007, Virology; 362:139-150). It is thuspossible to express known tumor-specific killing agent such as MDA7 toenhance the killing of tumors in vivo by this oncolytic virus.

Because wt PIV5 did not induce apoptosis in MDA-MB-435 cells, theeffective killing of tumors by wt PIV5 in vivo was puzzling. Possibly,PIV5 infection induced an innate immune response in nude mice, resultingin large number of monocytes in the tumors which would lead to celllysis. However, surprisingly, no abnormal infiltration of immune cellswas observed in the tumors. However, syncytia formation in PIV5 infectedtumor cells was observed in tissue culture and in tumors collected fromanimals after injection, suggesting syncytial formation may contributeto the killing of tumors. The oncolytic activity of mutant viruses withvarious mutations within the PIV5 proteins that enhance syncytialformation will be tested. Such mutants include, but are not limited to,those described by Paterson et al., 2000, Virology; 270:17-30.

Previously, a recombinant PIV5 with mutations at the V/P shared region(rPIV5-CPI−) was reported to inhibit tumor growth (Gainey et al., 2008,J Virol; 82:9369-9380). This recombinant virus was compared with wildtype PIV5 and wild type PIV5 was more effective than rPIV5-CPI− eventhough rPIV5-CPI− induced a severe cytopathic effect in infected cellscompared with wild type PIV5 in tissue culture (Wansley et al., 2003,Virology; 316:41-54). It is possible that PIV5 grows better in vivo thanrPIV5-CPI−, resulting in a more efficacious inhibition of tumor growthin vivo.

One of the main challenges for using oncolytic virus is that some spreadpoorly in vivo. This example is the first to demonstrate that PIV5replicated and spread in tumors in vivo, a characteristic that isessential for treating metastatic tumors, PIV5 appears to be safe inimmunodeficient mice since nude mice did not lose weight, show signs ofillness or show other pathological effects after tail vein injectionwith PIV5. The mechanism of this selectivity by an anti-tumor virus isnot clear. Interferon signaling plays a role in the selective oncolyticactivity (Stojdl et al., 2000, Nat Med; 6:821-825; Krishnamurthy et al.,2006, J Virol; 80:5145-5155; Obuchi et al., 2003, J Virol; 77:8843-8856;and Wollmann et al., 2007, J Virol; 81:1479-1491). However, since PIV5encodes the V protein that abrogates the interferon response, interferonsignaling may not play a major role in the selectivity of PIV5.

Recently, it has been reported that AKT, a host serine/threonine plays acritical role in replication of PIV5 (Sun et al., 2008, J Virol;82:105-114). It is thought that AKT1 phosphorylates the P protein ofPIV5, an essential co-factor of viral RNA polymerase, and that thisphosphorylation is important for the function of the P protein. AKT wasfirst discovered in retrovirus AKT8 as a viral proto-oncogene capable oftransforming certain cells (reviewed in Brazil and Hemmings, 2001,Trends Biochem Sci; 26:657-664). AKT is a key regulator in the PI3Ksignaling pathway, and plays an important role in many cellularprocesses such as cell survival, metabolism, growth, proliferation andmobility. AKT has been found to be activated in many cancers (Redaelliet al. 2006, Mini Rev Med Chem; 6:1127-1136; and Yoeli-Lerner and Toker,2006, Cell Cycle; 5:603-605). It has been reported that AKT1 plays acritical role in breast cancer (Ju et al., 2007, Proc Natl Acad Sci USA;104:7438-7443; and Sun et al., 2001, Am J Pathol; 159:431-437). Based onthe results that AKT, a kinase that plays a critical role in cancerdevelopment, also plays a critical role in the replication of PIV5, wehypothesize that AKT activation contributes to the selectivity of PIV5.Further testing of the role of AKT may reveal a novel pathway that iscritical for selectivity of oncolytic virus.

PIV5 selectively reduced the size of established tumors of MDA-MB-435human breast cancer cells and UACC903 human melanoma cells in a mousexenograft model. This negative strand RNA virus spread within the tumorand to non-adjacent tumors. It did not appear to infect normal mammaryepithelial cells and was not toxic to the animal. Thus PIV5 may bedeveloped as an alternative therapy for treatment of late stage and evenmetastatic breast cancer and other cancers.

Example 2 PIV5 as an Oncolytic Agent

Generation of recombinant PIV5 viruses that induce cell death in cancercells. To study the functions of the SH protein, a mutant PIV5 viruslacking SH (rPIV5ΔSH) was generated. The mutant virus induces increasedexpression of TNF-β in infected L929 cells and causes apoptosis.Addition of neutralizing antibody against TNF-β in the media ofrPIV5ΔSH-infected L929 cells blocks rPIV5ΔSH-induced apoptosis,indicating TNF-β plays an essential role in rPIV5ΔSH-induced apoptosis.To study the functions of the V protein, a mutant PIV5 lacking theconserved C terminus (rPIV5VΔC) was generated. Infection of rPIV5VΔCinduces increased expression of IL-6, IFN-β and enhanced IFN signalingin infected cells. Interestingly, while rPIV5VΔC induces apoptosis inHeLa cells, it does so through an intrinsic apoptotic pathway,independent of IFN. It is thought that rPIV5VΔC induces apoptosisthrough activation of Erstress initiated intrinsic apoptotic pathway.Mutant PIV5 viruses with mutations in the V/P gene such as rPIV5-CPI−and rPIV5-CPI+ that can also induce apoptosis in cancer cells have beengenerated. See, for example, He et al., 2001, J Virol; 75:4068-4079; Heet al., 1998, Virology; 250:30-40; Lin et al., 2003, J Virol;77:3371-338; Sun et al., 2004, J Virol; 78:5068-5078; Wansley et al.,2003, Virology; 316:41-54; Lin et al., 2007, Virology; 368(2):262-72;and Timani et al., 2008, Virol; 82:9123-9133. In addition, PIV5 mutantswith mutations in P and L that induce cell death in infected tumor cellsin vitro have been generated.

PIV5 and various mutant PIV5 were used to infect the well-studied humanmetastatic breast cancer cell line MDA-MB-231, in vitro. These cellswere derived from the pleural effusions of patients with metastaticbreast cancer (Welch, 1997, Clin Exp Metastasis; 15:272-306 and Welch etal., 2000, Breast Cancer Res; 2:408-416). PIV5, rPIV5VΔSH, rPIV5VΔC,rPIV5-CPI− and rPIV5-Rev were tested. PIV5 and all PIV5 mutants testedkilled the MDA-MB-231 cells, with rPIV5VΔC, rPIV5-CPI− and rPIV5-Revbeing most effective. Interestingly, killing of the cells by rPIV5-Revappears different from the killing by rPIV5VΔC and rPIV5-CPI− as themorphologies of the infected cells look different.

Efficacy of PIV5 mutant as an oncolytic agent against breast cancer in anude mouse model. As discussed in the previous paragraph, mutantrPIV5-CPI− was effective in killing breast cancer cells MDA-MB-231 invitro. To test the efficacy of the rPIV5-CPI virus in killing breastcancer cells in vivo, MDA-MB-231 were injected subcutaneously at theright flank of nude mice. Briefly, breast cancer cells MDA-MB-231 (10⁶cells) were injected into 4 to 6-week old nude mice subcutaneously. Atabout 8 weeks after the cancer cell injection, mice were injected withPBS, wild type PIV5 or the rPIV5-CPI− (CPI) in situ. PBS, PIV5 (10⁸plaque forming unit, pfu) or rPIV5-CPI− (CPI) (10⁸ pfu) were directlyinjected into tumors twice at one week interval. The growth of thetumors was monitored (tumor volume (length×width²/2) was monitored).Tumor growth rate is ratio of tumor volume at 4 weeks after infection tothe volume at the first injection. N=8 for each group. The sizes of thetumors at 4 weeks after the injection with rPIV5-CPI− were smaller thanthat of PBS or wild type PIV5-injected tumors, indicating that therPIV5-CPI− virus is effective in inhibiting the growth of breast cancercells in vivo.

Replication of PIV5 in tumors in vivo. To examine whether viruses werereplicating in cancer cells in vivo, the tumors were infected with PIV5and collected. Existence of the virus in the tumor was firstinvestigated using RT-PCR. RT-PCR reactions using primers specific forPIV5 genome or mRNA were carried out using tumors collected from theanimals. Viral transcripts and viral genomes were detected in the tumorsat 6 weeks after initial injection, indicating the virus was able toreplicate in cancer cells in vivo. Because PIV5 is a paramyxovirus, anegative stranded RNA virus, its life cycle does not have a DNA phase,and it is not known to cause latent infection. Hence, detection of viralgenomic (negative sense) RNA and mRNA normally indicates virusreplication. To further prove that virus replicates in tumors in vivo,virus titers were measured directly from extracted tumors using plaqueassay. MDA-MB-231 cells were injected into nude mice and waited forabout 6 weeks for tumors to develop. Then, PIV5 was injected into tumorsand at 1, 2 and 6 days after injection, tumors collected and the tumortissues homogenized. Virus titers were measured in the homogenizedtissues directly using plaque assay. Virus replication was detected intumors. Interestingly, there was an increase in virus titers between 2days post infection (dpi) and 1 dpi, indicating virus indeed replicatesand grows in tumors in vivo. The trend of virus growth continues to 6dpi, the last time point in the experiment, confirming that virusreplicates in tumors in vivo. This result is consistent with previousresults that viral genome RNA and viral mRNA were detected in tumorsextracted from animals. Thus, PIV5 replicates in MDA-MB-231 tumor cellsin vivo.

Toxicity of PIV5 in mice. To evaluate toxicity of PIV5 in nude mice,PBS, PIV5 or the rPIV5-CPI− virus were injected into nude mice throughtail veins. Briefly, nude mice were injected with PBS, PIV5 (10⁶ pfu) orrPIV5-CPI− (CPI, 10⁶ pfu) through tail vein (N=12 for each group). Themice were monitored for weight loss and signs of illness. Injections ofviruses did not cause weight loss or illness in nude mice up to 21 dayspost infection. For rPIV5-CPI− virus, the weight was followed up to 10weeks after injection and no sign of illness or weight loss were found.Lung, liver, heart, kidney and spleen of the mice were collected at 3,6, 10, 14, 21 days and 10 weeks after tail vein injections. The organswere sectioned and subjected to H & E staining. A board certifiedveterinary pathologist examined the slides blindly and found nosignificant lesions in all the samples, indicating PIV5 is safe to thenude mice.

As described in more detail in Example 1, MDA-MB-435 cells, instead ofMDA-MB-231 cells, were injected with wild type PIV5 virus. PIV5 iseffective in reducing tumor growth in vivo. Interestingly, the lowestdosage used (5×10⁴ pfu) was as effective as the highest dosage used(5×10⁶ pfu).

Effect of PIV5 on tumor in vivo. Because wild type (wt) PIV5 does notinduce apoptosis in MDA-MB-435 cells, the effective killing of tumors bywt PIV5 in vivo was puzzling. To determine if a PIV5 infection inducedmassive innate immune responses in nude mice, resulting in large numberof lymphocytes in the tumors and the immune responses killed tumors,tumors were collected after PIV5 or PBS injection, sectioned, andsubjected to H&E staining. Surprisingly, no abnormal infiltration ofimmune cells was observed in tumors. While some necrosis of cells in thecenter of tumors were observed in both PIV5 and PBS infected tumors,likely due to insufficient supply of nutrients to sustain rapid tumorgrowth, syncytia formations were observed only in PIV5 injected cells,suggesting that PIV5 may kill tumor cells by forming syncytia. Itappeared that tumor cells were being replaced by connective tissue,especially in the PIV5 sections.

Examination of PIV5 replication in vivo using live imaging. To measurevirus replication in tumors in vivo readily and without killing animals,a recombinant PIV5 that contains renilla luciferase as an extra genebetween the HN and L gene (rPIV5-RL) was generated. To examine whetherluciferase activity of infected cells reflects rate of virus infection,rPIV5-RL viruses after a series dilution (at factor 2) were used toinfect HeLa cells in 96-well plate. At 1 day post infection (dpi), thecells were lysed and processed for renilla luciferase activity accordingto manufacturer's instruction (Promega Inc., Madison, Wis.). The resultsindicate that at range of 0.06 to 8 MOI (multiplicity of infectivity,i.e., number of infectious virus per cell), the luciferase activitieswere in direct correlation with amount of viruses used, indicating thevirus can be used as an indicator for examining virus replicationquantitatively.

To examine replication of rPIV5-RL in tumors in vivo, the tumors wereinfected with rPIV5-RL and collected at 1, 3 and 6 days after injection.The tumors were homogenized and used for luciferase assay. Luciferaseactivity was detected, indicating that PIV5 replicates in tumors invivo. To examine replication of PIV5 in tumors in vivo without killingthe animals, an IVIS camera was used. The tumors were injected with6×10⁵ plaque-forming units (pfu) of rPIV5-RL. At 1 and 3 days aftervirus injection, substrate was injected into tumors directly andexpression levels of luciferase in tumors in mice were recorded usingthe IVIS camera. Luciferase activities were observed inrPIV5-RL-injected tumors, confirming that PIV5 replicates in tumors invivo in live animals. Interestingly, there are increases of theluciferase intensity over time within the same tumor, suggesting thatvirus may have spread to other cancer cells within the given tumors.Interestingly, no luciferase activity was observed in normal mousecells, suggesting that rPIV5-RL preferentially.

Spread of PIV5 in tumors in vivo. The ability to spread in vivo is acritical characteristic of any effective oncolytic virus, especially totarget metastatic cancer. To further test whether PIV5 can spread toareas beyond the original injection site, mice with two tumors wereused. Virus was injected in one tumor and whether there is expression ofluciferase in another tumor was examined. Expression of luciferase wasobserved in the tumor that was not injected with virus, indicating thatPIV5 can spread from the original site of injection to other sites.However, this experiment is that we cannot differentiate whether theluciferase activity in the other site is due to virus spread or virusspill during initial virus injection. It is possible that virus was notrestricted to the initial injection site during initial virus injectionand some entered the bloodstream and spread to the other tumor that wasnot injected with virus. To differentiate the difference between virusfrom initial injection and virus produced from the initially injectedtumor, a different injection regimen was used. Virus was injected intotumors as before. Then, 24 hours was to pass, to allow PIV5 that is notinside cells to dissipate. Then tumor cells were injected into the micethat have tumors that were already injected with PIV5. If virusreplication is observed in these newly injected cells, the virus willlikely come from the tumors that are injected with virus, indicatingthat virus can spread from tumors to other tumor cells. Following thenew regimen of virus and cells injection, expression of luciferase wasobserved in the newly injected cells, indicating that PIV5 spreads toother sites in vivo. Using both 5 mm and 10 mm distance (distancebetween tumors injected with virus and site of cell injection), it wasfound that both cells expressed luciferase, indicating that PIV5 canspread at least 10 mm in vivo. Interestingly, luciferase activity wasonly observed in tumor cells, but not in normal mouse cells, suggestingthat PIV5 preferentially infects tumor cells in vivo and/orpreferentially replicates in cancer cells over normal cells. The abilityto spread within a host is critical for oncolytic virus to be used as ananti-cancer agent. This has been a challenge for adenovirus-basedoncolytic virus studies. Examples 1 and 2 indicate that PIV5 spreads intumors in vivo. PIV5 will be tested against metastasized breast cancerin vivo in Example 3.

Efficacy of PIV5 mutant as an oncolytic agent against lung cancer in asyngeneic mouse model. To investigate whether PIV5 mutants have thepotential to reduce the size of solid tumors in an immune competentanimal, a tumor model system based on C57BL/6 mouse injected withtumorigenic Lewis lung carcinoma cells (LL2) (Bertram and Janik, 1980,Cancer Lett; 11) will be used. To start this study, the killing of theLL2 cells by PIV5 was examined in vitro and it was found that rPIV5-CPI−induces CPE. Briefly, mouse lung cancer cells (LL2) were injected intoC57/B16 mice. The tumors developed from the cells were injected withrPIV5-RL, a recombinant PIV5 containing luciferase gene. The tumors fromthree mice were collected at 1, 2, 3 and 6 days after virus injectionand processed for luciferase assay. Then, cells were injectedsubcutaneously in the right flank of C57/B16 mice. Then PBS, wild typePIV5 and the CPI− virus were injected into the mice through tail veins.Briefly, C57/Black 6 mice were injected with lung cancer cells LL2. PIV5(4×10⁶ pfu), CPI (4×10⁶ pfu) or PBS was injected through tail veins ofthe mice (n=8 for each group) at three days intervals. Tumor growth andweight of mouse were monitored. The experiment was terminated at day 14after injection of the virus to relieve the pain and suffering of themice due to the over-growth of the tumors in the control group followingthe instructions from our IACUC. Tumor size as well as weight wasmonitored. It was found that the CPI− virus injection reduced the tumorsizes by average about 50% and no differences in the weights of the micewere found.

Thus, testing the oncolytic activity of rPIV5-CPI− in vivo indicatesthat rPIV5-CPI− is effective in reducing tumor growth in both a nudemouse model and in a C57/B16 mouse model. Replication of PIV5 in tumorsin C57/B16 mice. To investigate whether PIV5 replicates in tumors invivo, rPIV5-RL was injected into tumors from lung cancer cells in vivoand measured luciferase activity of the tumors at various points aftervirus injection. Luciferase activity was detected in tumors at 1, 2, 3and 6 days after injection, indicating that PIV5 replicates in tumors invivo. An increase of luciferase activity was also observed at day 2,indicating virus growth since viral protein production reaches a peak at24 hours post infection. Interestingly, luciferase activities were alsodetected in the spleens of mice even though the virus was injected intotumors directly.

The efficacy of rPIV5VΔC as an oncolytic agent was tested. While PIV5has oncolytic activity, the rPIV5VΔC mutant construct was tested, to seeif oncolytic activity can be enhanced. rPIV5VΔC, a recombinant PIV5lacking the conserved cysteine rich C terminus of the V protein, inducesapoptosis in infected cells (He et al., 2002, Virology; 303:15-32).While average tumor size of PBS-treated group reached 1500 mm³, an upperlimit of tumor size allowed by IACUC guideline, average sizes of PIV5and rPIV5VΔC infected tumors were about 400 mm³ and 200 mm³ respectivelyin 5 weeks, indicating both viruses are effective against tumor growthin vivo. Interestingly, half of the tumors (4 out of 8) disappeared inrPIV5VΔC-infected group, 1 out of 8 in PIV5 group and none in PBS group.Statistical analysis shows the difference between PIV5 and rPIV5VΔCtreatment is statistically significant. Thus, rPIV5VΔC is more effectivethan PIV5.

Both rPIV5VΔC and rPIV5-CPI− induced cell death on infection of breastcancer cells such MDA-MB-231 and MDA-MB-435 and lung cancer cells suchLLC2. Additional PIV5 mutants will be tested to identify mutations andcharacteristics that will further enhance oncolytic activity of PIV5.

Generation of rPIV5-MDA7 and preliminary testing of rPIV5-MDA7 in vivo.A recombinant PIV5 containing a MDA7 gene between the PIV5 HN and Lgenes (rPIV5-MDA7) has been produced. RT-PCR was performed to confirminsertion of the MDA7 gene between the HN and L genes in putativerPIV5-MDA7-infected cells using primers specific to the HN and L genesand the resultant RT-PCR fragment sequenced. The sequence from theRT-PCR product match the input cDNA sequence, indicating rPIV5-MDA7 hasbeen obtained.

Briefly, for the testing of rPIV5-MDA7 and rPIV5-Bax, breast cancertumors were developed as previously described herein. Each tumorreceived 50 μl of PBS, or a dose of 1×10⁶ pfu virus in 50 μl volume.Injection was repeated at an interval of one week. N=6 for each group.The tumor sizes (width²×length/2) for PBS and rPIV5-MDA7 injections werebetween 120-200 mm³. Each tumor received 50 μl of PBS, or a dose of1×10⁶ pfu virus. Injection repeated at an interval of one week. N=9 forPBS and rPIV5-MDA7 group; N=6 for rPIV5-Bax. rPIV5-Bax injection dosewas 2.5×10⁴ pfu. At 5 weeks after PBS injection, the PBS group wasremoved from the experiment because tumors became too large. However,observation of tumors injected with viruses continued. By 8 weeks aftervirus injection, all tumors disappeared from rPIV5-MDA injection group.P<0.005 for all time points between PBS and rPIV5-MDA7.

The rPIV5-MDA7 virus replicates as well as wild type and its efficacy ininhibiting tumor growth has been tested. MDA-MB-435 cells were injectedinto nude mice and then injected PBS, PIV5 (wild type, wt) or rPIV5-MDA7into the tumors directly. Amazingly, rPIV5-MDA7 reduced size of tumorsdramatically. As described in Example 1, with the mutant rPIV5-CPIvirus, inhibition of tumor growth was only observed after injection ofa, i.e., the tumors continued to grow after the virus injection but at aslower rate. With rPIV5-MDA7, the size of tumors injected with actuallyreduced. Not only the actual size of tumor was reduced, dosage of virusthat is effective was much lower than before (only 1×10⁶ pfu ofrPIV5-MDA7 vs. 1×10⁸ pfu of rPIV5-CPI− for each injection). Theseresults have been confirmed.

In addition, a recombinant virus expressing Bax (rPIV5-Bax) has beentested, which is thought to be one of the strongest cell death inducers.Preliminarily, rPIV5-Bax was not as effective as rPIV5-MDA7 even thoughrPIV5-Bax inhibited tumor growth initially.

Mutants such as rPIV5VΔC and rPIV5-CPI− (or other mutations withincreased oncolytic potential) and expression of MDA7 will be combinedtogether to generate a new recombinant PIV5 and to test its efficacy asdetailed in Example 4. In addition, a direct comparison of efficacy ofrPIV5-MDA7 to AdV-MDA7 (an oncolytic virus that is in clinical trial)will be undertaken.

AKT plays a critical role in replication of PIV5 and other negativestranded RNA viruses. Non-segmented negative stranded RNA viruses suchas measles virus (MeV), Newcastle disease virus and vesicular stomatitisvirus (VSV) have been used as oncolytic agents and some of them haveadvanced to clinical trials. However, the underlying reason that causesthese viruses to preferentially replicate in cancer cells is not clear.We have found that siRNA and inhibitors against AKT1, a kinase thatplays an important role in cancer development, block replication of PIV5as judged by viral protein expression and virus growth, as well as MeVand VSV. Since AKT is often activated in cancer cells, it is possiblethat the oncolytic viruses from PIV5, MeV or VSV target cancer cellsbecause of their dependence on AKT for replication, providing anexplanation for the selectivity of oncolytic viruses on cancer cells.

A role of AKT1 in phosphorylation of the P protein of PIV5. In theexperiments described above, inhibitors were added after virus wasincubated with cells, indicating the effect of the inhibitors on thevirus life cycle was post-entry. The fact that the siRNA against AKT andthe inhibitors against AKT reduce viral protein expression indicatesthat AKT likely plays a role in viral RNA synthesis. To examine thispossibility, a PIV5 mini-genome construct was utilized (Lin et al.,2005, Virology; 338:270-280). AKT inhibitor blocked reporter geneexpression from the mini-genome system, indicating that AKT plays acritical role in viral RNA synthesis (Sun et al. 2008, J Virol;82:105-114). The fact that AKT inhibitor inhibits viral RNA synthesisfrom the mini-genome system, suggests that AKT likely targets acomponent of the viral RNA polymerase complex, which is minimallycomprised of the P and L proteins for all NNSVs. It is known that thephosphorylation of the P protein plays a critical role in viral RNAsynthesis (Lu et al., 2002, J Virol; 76:10776-10784). Since AKT is aprotein kinase, the effect of the AKT inhibitor on phosphorylation of Pin PIV5-infected cells was examined. The AKT inhibitor reducedphosphorylation of the P protein of PIV5 by about 30% without affectingviral protein translation during the same labeling period. Theincomplete inhibition of P phosphorylation by AKT inhibitor is likelydue to the fact that P has multiple phosphorylation sites and AKTcontributes to phosphorylation of some of the sites (or a single site).

The fact that AKT inhibitor reduces phosphorylation of P indicates AKTplays a role in P phosphorylation. However, it is not clear whether theeffect is direct or indirect. To test whether AKT can phosphorylate Pdirectly, PIV5 P protein was purified from bacteria using a His-taggedvision of the P protein using an approach similar to the ones employedto purify large numbers of bacteriophage T7 RNA polymerase (He et al.,1997, Protein Expr Purif; 9:142-151). In vitro kinase assays were beencarried out and it was demonstrated that AKT1 phosphorylates therecombinant P protein from bacteria. To ensure the proteinphosphorylated by AKT is indeed P, not a non-specific bacteria proteinco-purified with P and migrated at the same location as the P protein,the recombinant P protein was further immunoprecipitated with P-specificantibody (P282 and Pk) and carried out the in vitro kinase assay. Theresult confirms that the recombinant P purified from bacteria isphosphorylated by AKT1. These data strongly indicate that AKTphosphorylates P in infected cells directly and this phosphorylationplays a critical role in the function of the P protein. To furtherexplore this, the phosphorylation site of AKT within P will beidentified.

Example 3 Phase I/II Clinical Trial of Canine Parainfluenza Virus as anAnti-Cancer Agent

Cancer causes significant morbidity and mortality in both dogs andpeople. Surgery, radiation, and chemotherapy can be used to treat andcontrol some cancers; however, these approaches are often ineffectiveagainst late to end stage disease. In addition, removal of tumors isinvasive, and radiation and chemotherapy carry risk of severe, unwantedside effects. Safer, more effective treatments are needed.

The use of these viruses as anti-tumor agents is being pursued as analternative to standard cancer therapies. Many viruses have demonstratedefficacy in killing cancer cells in vitro and in vivo and some arecurrently being tested in human clinical trials. For example, ONYX-015,an adenovirus derived anti-tumor agent, has been used alone and incombination with chemotherapy in clinical trials for a variety of humancancers. Phase I/II trials of ONYX-015 in treating head and neck cancerhave demonstrated that it is safe and effective. PV701, a viralanti-tumor agent developed from Newcastle disease virus (NDV), has beenevaluated in human Phase I trials to treat cancer patients with advancedsolid tumors including melanoma, and cancers of the head and neck,colon, and pancreas. Initial results are promising, and additionaltrials sponsored by the National Cancer Institute are ongoing. However,only a few of the many, many known viruses have shown promise in cancertreatment.

The previous examples determined that canine parainfluenza virus (CPI;also known as parainfluenza virus 5 or PIV5) is effective in treatinghuman breast cancer, human melanoma, and human lung cancer using mousexenograft models. CPI is an RNA virus in the family Paramyxoviridae. Asan RNA virus, it is safer for use in cancer patients than DNA viruses(such as adenovirus) because it does not have a DNA phase in its lifecycle. As such, using CPI avoids the possible unintended consequences ofgenetic modifications of the host cell's DNA by viral DNA recombinationor insertion. RNA recombination has not yet been reported.

With this example, dogs with naturally occurring cancers will be used toevaluate the safety and overall response rate (ORR) of CPI-WT intumor-bearing dogs. This study will determine the dose-limitingtoxicity, maximally tolerated dosage, and potential efficacy of wildtype canine parainfluenza virus (CPI-WT) in dogs with malignant solidtumors. It will also determine the extent of CPI-WT viral shedding frompatients following intra-tumoral virus administration. Viral-basedtherapies, including recombinant viruses expressing tumor killingproteins (e.g. MDA7), will be studies as anti-cancer agents for pets,and eventually people.

Cancer is a leading cause of death in the adult pet population, andmalignant solid tumors (MST) encompass a large proportion of cancersdiagnosed. Sarcomas, carcinomas, and even some round cell tumors areincluded in this classification. Although certain types of MST occurmore commonly in specific breeds (e.g. histiocytic sarcoma in theBernese mountain dog and flat-coat retriever), in reality, all dogs areat risk of MST development. Currently available treatment modalities fordogs with MST include surgery, radiation therapy, and chemotherapy.These treatments are invasive, carry risk of morbidity, are costly, andin many cases are palliative, not curative. Thus, search for efficaciousand cost-effective therapies is constant.

Comparative oncology is a discipline that merges the study of naturallyoccurring cancers in pet animals with studies of cancers in people. Petdogs are practical models because their cancers arise spontaneously, inanimals with intact immune systems. Furthermore, many canine cancersmore accurately emulate human cancers in etiology, behavior, treatment,and outcome. This example will provide an additional model system fordeveloping novel oncolytic viral therapies for the treatment humans withcancer.

Malignant solid tumors are neoplastic masses of abnormal tissue thatexhibit invasion into surrounding normal tissues, and in some cases havehigh metastatic potential. MST most commonly arises from mesenchymal(sarcomas) or epithelial tissues (carcinomas) and can occur almostanywhere in the body. Treatment, including surgery, radiation, andchemotherapy, is invasive and carries risk of significant toxicity.These treatments are costly, and for many dogs, they do not result incure.

Canine parainfluenza virus (also known as parainfluenza virus 5 or PIV5outside of veterinary field) is an RNA virus in the familyParamyxoviridae. As an RNA virus, it is seemingly safer for use incancer patients than DNA viruses (e.g. adenovirus) because it does nothave a DNA phase in its life cycle. As such, using CPI avoids thepossible unintended consequences of genetic modifications of the hostcell's DNA by viral DNA recombination or insertion. RNA recombinationhas not yet been reported. CPI can infect many cell types with littlecytopathic effect. However, in some cell types, CPI infection causessyncytia formation leading to cell death. The previous examples havedemonstrated that CPI infection reduces tumor growth in vivo, likelythrough syncytia formation.

Although CPI was originally isolated from cultured primary monkey cells,its natural host is the dog. In most dogs, CPI infection causes no overtclinical signs of disease. Furthermore, most dogs have been vaccinatedagainst this virus. When disease occurs, it manifests as an upperrespiratory tract infection (sneezing, nasal discharge, and occasionallycough). With supportive care, prognosis is excellent. Based on itssafety record, CPI is an attractive oncolytic virus therapy in dogs.

Trial Design

Inclusion Criteria. Dogs with histologically confirmed spontaneous MSTamenable to repeated incisional biopsies are eligible for inclusion.Dogs must be otherwise healthy.

All owners are required to sign an informed consent form prior toentering his/her dog into the trial.

Treatment, Assessment and Follow-up. To investigate the objective ofmaximum tolerated dosage and associated treatment toxicity, aconventional 3+3 phase I trial design will be used, enrolling patientsin cohorts of 3. The first cohort will be treated at 1×10⁸ pfu, anddosage escalations will occur in 1×10¹ pfu increments. All toxicitieswill be defined using the Veterinary Co-operative Oncology Group CommonTerminology Criteria for Adverse Events (VCOG-CTCAE) v1.1. Unacceptablegrade of toxicity will be dependent upon category of toxicityexperienced. If unacceptable toxicity occurs in one of the three dogs inany cohort, an additional three dogs will be enrolled in that cohort.The goal will be to establish the maximum tolerated dosage (the dosageat which approximately 33% of patients experience unacceptable toxicity)after treating at least 6 dogs in the cohort where toxicity occurs.Overall patient health and occurrence of adverse events will be assessedby patient history and physical examination prior to treatment and ateach visit. A complete blood count, serum biochemical profile, andurinalysis will be performed prior to the first treatment and will berepeated prior to the 4th treatment and one month after treatmentcompletion.

A maximum tolerated dosage may not become evident for virus-basedtherapies as it does for traditional cytotoxic chemotherapeutics. If so,patient accrual could continue indefinitely. Therefore, as an additionalendpoint, tumor responses will be evaluated via primary tumormeasurements. Antitumor responses will be assessed according to ResponseEvaluation Criteria in Solid Tumors (RECIST). The longest diameter ofthe tumor is recorded and evaluated at each examination. Completeresponse (CR) is defined as disappearance of the tumor or all measurablelesions. Partial response (PR) is defined as at least a 30% decrease inthe longest diameter of the tumor or the sum of the longest diameters ofthe measurable lesions. Progressive disease (PD) is defined as a 20%increase in the longest diameter of the tumor or the sum of the longestdiameters of the measurable lesions or the appearance of new lesions.Stable disease (SD) is defined as insufficient shrinkage to qualify forPR and an insufficient increase to qualify as PD. Overall response rate(ORR) will include patients experiencing CR or PR.

The intent will be to administer four weekly doses of virus to each dogin the study. The calculated dose will be prepared in a volume of 0.5 to1.0 mL. Virus will be administered intratumorally into four quadrants ofthe tumor. Tumor biopsies will be performed prior to treatments #1 and#4, as well as 4 weeks after treatment completion. Biopsies will beevaluated histopathologically for evidence of regression and to confirmviral infection in tumor.

Assessment of CPI-WT Shedding. Nasal swabs and fecal and blood sampleswill be collected from dogs prior to treatment initiation, at the timeof each weekly treatment, and 2 and 4 weeks following treatmentcompletion. Samples will first be analyzed using RT-PCR. If viral RNA isdetected, the numbers of live virus in the samples will be determined.

For this phase I trial design, no special statistical calculations arerequired. The target enrollment for response assessment is determinedusing the Simon Minimax design, with a rule-out response probability of5%, a minimum useful response probability of 25%, an α of 0.05 and a βof 0.10. Based on this design, 15 dogs will be enrolled in the firststage of this trial. If no responses are seen in the first 15 patientsentered, the trial may be terminated. If a promising response rate isnoted, 5 additional dogs will be enrolled with residual funding of thisproposal, and the trial will be continued as part of a competitiverenewal proposal.

Because most dogs have been vaccinated against CPI, immunity to CPI maypose a problem. As demonstrated in the other examples included herewith,CPI-positive dogs have been successfully immunized against influenzavirus using a CPI-based vaccine. Based on these results, CPI will likelyinfect solid tumors in previously vaccinated dogs as well.

This is the first oncolytic virus clinical trial in dogs, and it willprovide valuable information about the safety and efficacy of CPI-basedcancer therapy as an alternative to current cancer treatments. Based onthe previous examples, it is expected that the intra-tumoraladministration of CPI will be safe and will reduce the size of tumors.Furthermore, significant viral shedding from intra-tumorally treateddogs is not expected, as virus was not detected in nasal swabs of dogsinfected intranasally or intramuscularly with this respiratory virus.

The studies described above will be repeated with mutant viralconstruct, such as a PIV5 viral construct altered to incorporate tumorkilling genes such as MDA-7, potentially boosting efficacy.Additionally, these virus-based can be incorporated into other cancertreatment protocols, in hopes of improving tumor control, extendingsurvival times, and possibly improving quality of life. Alternatively,virus-based treatments can be used as a sole modality in cases whereother treatments are not viable options.

Success of the proposed experiments will not only lead to a noveltherapy for dogs with cancer, but also will move the oncolytic virusfield forward. If dogs with spontaneous tumors are established as asuitable model for human cancer treatment with oncolytic viruses, moreresearch will likely be performed, thereby potentially leading to morecancer treatment options for dogs and people.

Five client-owned dogs with either melanoma or sarcoma have been tested.Injections of 1.6×10⁸ pfu to 3.2×10⁸ pfu PIV5 have been well-toleratedby the dogs. No clinical illness has been observed to associate with theinjections, indicating that PIV5 injection is safe in dogs.

Example 4 Infection of Dogs with Prior Exposure to PIV5

Infection of “naive” dogs with PIV5 and rPIV5-H3. Dogs were inoculatedwith rPIV5-H3 via intranasal route, and determined replication of virusin dogs and measured immune responses to the virus. Dogs are routinelyvaccinated with vaccines containing live PIV5 at a young age (as earlyas 3-week old). Through an arrangement with the animal vendor, 8 dogs at12-week of age without vaccination of live PIV5 were obtained. Thetiters of PIV5 antibodies in these dogs were determined using ELISA andneutralization assay. All dogs were positive to PIV5 in ELISA. However,neutralization antibody (nAb) titers were undetectable. The dogs (n=4)were infected with PIV5 or rPIV5-H3 via intranasal (IN) route. At 3 and5 days post infection, nasal swabs were taken from infected dogs, andassayed for existence of viruses. While no virus was detected when theswabs were analyzed using plaque assay, RT-PCR products were detected in7 of 8 dogs at 3 days post infection (dpi) and very weak RT-PCR signalswere detected in 5 of 8 dogs at 5 dpi, suggesting that limitedreplication of PIV5 in naris of infected dogs at 3 days post-infectionand the infection was being cleared at 5 days post-infection. The dogswere bled at 21 days post infection. Increases in anti-PIV5 titers weredetected in all dogs, suggesting that the dogs were infected.Measurement of anti-HA titers using HAI assay indicated that allrPIV5-H3 inoculated dogs seroconverted and had HAI titers at average42.5 (range from 20 to 80) at 3-week post-infection. No HAI was detectedin dogs-inoculated with PIV5.

Infection of dogs with exposure to PIV5 with PIV5-HA. To examine whetherdogs with prior exposure with PIV5 can still be vaccinated withrecombinant PIV5-based vaccines, dogs were obtained that were vaccinatedagainst PIV5 multiple times and had anti-PIV5 neutralizing antibodies.The dogs were infected with rPIV5-H3 via IN route. No virus was detectedusing plaque assay at 3 and 5 dpi in naris of infected dogs. One out ofeight dogs tested positive using RT-PCR at 3 dpi. Dogs were then bled at3 weeks post-infection. The dogs vaccinated with rPIV5-H3 had HAI titersranging from 40 to 80 (average 77, 1 at 40 and 5 at 80), indicating thatrPIV5-H3 vaccination generated immunity against influenza virus (a4-fold increase of HAI titer or a HAI titer of 40 is consideredprotective against influenza virus infection). The nAb titers againstPIV5 also increased in rPIV5-H3-infected dogs, confirming the infectionof the dogs with rPIV5-H3.

The nAb titers against PIV5 in vaccinated dogs were higher than the“naive” dogs and were as high as 300 (FIG. 14B). All dogs with nAbagainst PIV5 seroconverted after a single dose IN inoculation ofrPIV5-H3, and the titers of anti-H3 antibody had no correlation to thenAb titters against PIV5, further confirming that nAbs of PIV5 had nopredictive value in determining immune responses to a PIV5-based vaccinein dogs. The highest titer of nAb against PIV5 in humans is 60, lowerthan the titers of nAb against PIV5 in dogs. Thus, neutralizing antibodyagainst PIV5 in humans will likely not prevent PIV5-based vaccinecandidates from generating protective immunity.

This Example has published as Chen et al., “Evaluating a ParainfluenzaVirus 5-Based Vaccine in a Host with Pre-Existing Immunity againstParainfluenza Virus 5,” PLoS One; 2012;7(11):e50144, doi:10.1371/journal.pone.0050144, Epub 2012 Nov. 20, which is incorporatedby reference herein in its entirety.

Example 5 PIV5 Mutants as Anti-Tumor Agents for Metastatic Breast Cancer

The previous examples demonstrate the oncolytic activity of PIV5 andPIV5 mutants, showing that PIV5 has oncolytic activities against solidtumors in a breast cancer model using nude mice, a melanoma cancer modeland a lung cancer model using C57/B16 mice. Importantly, these previousexamples also demonstrated that PIV5 can spread from cancer cells tocancer cells in vivo. This example will continue to test PIV5 as anoncolytic agent against metastatic cancer.

PIV5 WT, rPIV5-CPI− and additional mutant PIV5 virus mutants will betested, following procedures described the previous examples, in thenude mice models including solid tumor model and metastatic model aswell as in an immune competent lung cancer model. The following will betested:

PIV5-WT;

rPIV5-CPI− (Wansley and Parks, 2002, J Virol; 76:10109-10121; and Timaniet al., 2008, Virol; 82:9123-9133), having Y26H, T32I, V33I, L50P, L102Pand S157F mutations of the V/P gene;

rPIV5-CPI+(Wansley and Parks, 2002, J Virol; 76:10109-10121; and Timaniet al., 2008, Virol; 82:9123-9133), having T32I, V33I and S157Fmutations of the V/P gene and demonstrating phenotypic properties ofinduction of CPE and apoptosis;

rPIV5-Rev, having a S1561 mutation of the V/P gene and demonstratingphenotypic properties of induction of CPE and apoptosis;

rPIV5ΔASH (Lin et al., 2003, J Virol; 77:3371-3383), having a deletionof the SH gene and demonstrating phenotypic properties of induction ofCPE and apoptosis;

rPIV5-P-S157A, having a S157A mutation of the V/P gene and demonstratingphenotypic properties of induction of CPE and apoptosis;

rPIV5-P-S308A, having a 5308A of the P gene and demonstrating phenotypicproperties of induction of CPE and apoptosis;

rPIV5-L-A1981D, having A1981D mutation of the L gene and demonstratingthe phenotypic property of induction of CPE; and

rPIV5-F-5443P (Paterson et al., 2000, Virology; 270:17-30), having aS443P mutation of the F gene and demonstrating the phenotypic propertyof induction of massive syncytia formation.

Further experiments will combine effective mutations within the PIV5genome and test its efficacy in vivo.

Testing efficacies of PIV5 mutants against solid tumors. The breastcancer model system in nude mice will be used to examine the effects ofthe additional oncolytic virus candidates such as rPIV5ΔASH, rPIV5-CPI+,rPIV5-Rev, rPIV5AC, rPIV5-P-S157A, rPIV5-P-S308A, rPIV5-L-A1981D andrPIV5-F-5443P on tumor growth in vivo. Recent studies of functions ofthe P protein show that mutating S157 of P to A or S308 to A results ina recombinant virus that induces cell death. Also, a mutation in the Lgene of PIV5 (rPIV5-L-A1981D) has been identified that causes celldeath. These cell death inducing mutants will likely enhance oncolyticactivity of PIV5. It has been reported before that mutation of S to P at443 position of the F protein causes the F protein to be more effectivein promoting cell-to cell fusion (Paterson et al., 2000, Virology;270:17-30). rPIV5-F-S443P, a recombinant virus promotes rapid syncytiaformation, may be more effective as an oncolytic virus comparing to wildtype PIV5.

Metastatic breast cancer cells MDA-MB-435 (Morales et al., 1999, NatGenet; 21:115-118; and Yang et al., 1999, J Biol Chem; 274:26141-26148)cells will be used. The cells will be grown in culture to subconfluence,released from the plate and concentrated to about 5×10⁵ cells in 200 μlof PBS for injection. For studies of the effects of virus on solidtumors, the cells will be injected into the right supra scapular area of5-6 week old female athymic mice subcutaneously in a group of 8-10 micefor each virus following well established procedures (Welch, 1997, ClinExp Metastasis; 15:272-306). The development of tumor will be monitoredby measuring the mean tumor diameter (the square root of the product oftwo orthogonal measurements). The tumors will also be examined byhistology to determine the status of the tumor cells and the surroundingmammary tissue. Tumors usually become visible by about 2 weeks and willbe fully developed in 4-5 weeks when the average of the size of tumorsis about 7 mm by 7 mm. At that time, equal numbers (10⁶ plaque formingunit, pfu) of purified PIV5 wild type, rPIV5ΔASH, rPIV5VΔC, rPIV5-CPI−,rPIV5-Rev rPIV5-P-S157A, rPIV5-P-S308A, rPIV5-LA1981D or rPIV5-F-S443Pin 50 μl of PBS or PBS will be injected into the tumor directly. Animalswill be monitored and the sizes of the tumors will be measured dailyafter administration of the virus. Distressed animals will besacrificed. The experiment will be terminated by about 60 days or soonerbefore the tumors reach 1.5 centimeter. If one specific mutant virus isfound to be more effective in reducing tumor growth, a serial dilutionof input virus will be used to determine the lowest dosage that iseffective in reducing tumor growth efficiently.

Testing efficacies of PIV5 mutants against metastatic cancer. In orderto study the effects of the virus on metastasis the cancer cells (forexample, MDA-435GFP, previously described) will be injected into thetail vein of mice with about 5×10⁵ metastatic breast cancer cells in 100μl of PBS. The animals will be monitored daily for the presence ofpalpable tumors. Distressed animals will be sacrificed. After 3 weekswhen animal normally develop metastatic cancer, the animals will beexamined for the presence of tumors in the lungs and lymph nodes andwild type PIV5, PIV5 mutants in 100 μl of PBS or PBS will also beinjected into the mice intravenously. The experiment will be terminatedby about 60 days or sooner when animals appear severely distressed.

If the virus has an effect on soft tissue metastatic, it will bedetermined if the virus affects metastasis to bone. This will be done byinoculation of the cancer cells (about 2-3×10⁵ cells in 200 μl PBS,MDA-435GFP) into the left ventricle of the heart of athymic mice. Thisprocedure leads to visible bone osteolysis in about 4-5 weeks afterinoculation. Then wild type PIV5, PIV5 mutants in 200 μl PBS or PBSalone will be injected into the animals intravenously. After 4 weeks thefemurs will be removed, fixed, decalcified, embedded and sectioned. Thesections will be stained with H&E for visualization of the cancer cellsand for condition of the bone as described before (Phadke et al., 2006,Clin Cancer Res; 12:1431-1440; Mastro et al., 2003, Clin Exp Metastasis;20:275-284; Mastro et al., 2004, J Cell Biochem; 91:265-276; and Harmset al., 2004, Clin Exp Metastasis; 21:119-128).

Bio-distribution of virus and tumor biology. Metastasis of the tumorsand localization of virus in vivo will be examined at 3, 7 and 14 dayspost infections. Initially, rPIV5-RL will be used to monitordistribution of PIV5 within mice. For PIV5 without luciferase gene,three mice will be sacrificed and organs including lung, liver, kidney,heart and brains collected. The collected organs will be frozen in−80-degree methyl butane and sectioned sagittally at 16 μm on acryostat. The sections will be fixed with acetone, washed with PBS andthen blocked in 3% normal goat serum in PBS for 30 minutes. To studymetastasis of the cancer cells, antibody against GFP will be used toincubate with sectioned organ slides; to study localization of thevirus, expression of PIV5 virus proteins will be used as an indicator.Appropriate secondary antibodies labeled with fluorescence materialssuch as PE or Texas Red will be used to incubate with the sections andresults will be examined using a confocal microscope as described before(Rubin et al., 2003, J Virol; 77:11616-11624).

Virus titers in organs including lung, liver, kidney and brain will beexamined. At 3, 7 and 14 days post incubation with viruses, 3 mice willbe sacrificed and the organs will be collected. The organs will beweighed and homogenized. Titers of viruses will be determined usingplaque assays as described in He et al. (He et al., 1997, Virology;237:249-260).

To study pathology and metastasis of the tumors, major organs includinglung, heart, kidney, liver and brain of the mice will be collected atthe time of experiment termination. Part of the samples will besectioned and analyzed as described above. Part of the organs will besubjected to pathology studies.

To investigate mechanism of oncolytic activity of PIV5 mutants and tomake PIV5 more efficient in reducing tumor growth in vivo, tumor biologyafter virus infection will be examined, as described earlier. Tumorswill be harvested, sectioned, stained and examined by a veterinarianwith training in rodent pathology. Tumor type and progression will beassessed including differentiation, presence of mitotic figures,necrosis, vascularity, and the presence or absence of inflammatoryinfiltrates and apoptotic bodies.

Since PIV5 causes syncytia in tumors, it is expected that tumorsinfected with PIV5 mutants will also show syncytia. For mutants thatinduce cell death, apoptotic cancer cells will also likely be observedin tumors infected with the viruses. In tumor sections from a pilotstudy, necrosis was present in both control and PIV5 infected sections,although more pronounced in the PIV5 sections. There was minimalinflammatory response, and no appreciable angiogenesis in any of thesections. Fibrosis and loose connective tissue were more prevalent inthe PIV5 infected sections, and syncytia were noted in 2 of 3 PIV5infected sections and 0 of 3 control sections.

It is possible that single mutations of PIV5 will not be effective inkilling metastatic breast cancer in vivo. A recombinant PIV5 with acombination of different mutations of PIV5 such as rPIV5ΔSH-CPI−, orrPIV5ΔSH-Rev will then be generated and tested using the sameexperimental regiment. The V protein of PIV5 plays an important role inevading host innate immune responses by causing degradation of STAT1, akey regulator of IFN signaling pathway. Interestingly, mutations in theV protein of rPIV5-CPI− render the V protein ineffective in blocking IFNsignaling (Wansley et al., 2005, Virology; 335:131-144; andChatziandreou et al., 2002, Virology; 293:234-242). It has been reportedthat rPIV5-CPI− replicates preferentially in cancer cells over normalcells in the presence of interferon, likely due to possible defectiveIFN signaling pathways often associated with cancer cells (Obuchi etal., 2003, J Virol; 77:8843-8856). The sensitivity of the virus to IFNcan provide additional selectivity for oncolytic virus. Similarly,rPIV5VΔC, which induces production of IFN-β and is unable to block IFNsignaling, may provide additional selectivity for oncolytic virus. Thus,incorporating these mutations (CPI− and/or VΔC of the V protein) willlikely increase selectivity of the oncolytic virus. If a recombinantPIV5 with a combination of different mutations is determined to be moreeffective, a luciferase gene can be inserted into the genome of thisvirus for bio-distribution study.

C57/B16/3LL model. To examine whether the viruses can kill tumors in animmune competent animal model system, the viruses will be injected intoC57BL/6 mice carrying solid tumors generated from Lewis lung carcinomacells (Bertram and Janik, 1980, Cancer Lett; 11:63-73). To obtainC57BL/6 mice with solid tumors, LL2 cells (5×10⁵ cells) will be injectedsubcutaneously (sc) in the right supra scapular area (Sharma et al.,1999, J Immunol; 163:5020-5028). Tumor growth will be monitored threetimes a week using a caliper. Once the tumor sizes reach to 8 mm indiameter (the larger diameter, not the smaller diameter), 10⁶ pfuviruses in up to 50 μl volume will be injected into the tumors directly.If needed, up to 10¹⁰ pfu may be used. The mice will be monitored dailyinitially. If the size of the tumor is not reduced in one week,additional injection of the viruses will be carried out. The experimentwill be terminated when the tumor reaches 15 mm in diameter (the largediameter). Localization and titers of viruses will be examined asdescribed above. In addition, samples of tumors and adjacent tissueswill be collected for histopathology studies and for examination ofpresence of virus as well as tumor biology and immune responses asdescribed in previous section. In additional experiments, a recombinantPIV5 with a combination of different mutations of PIV5 such asrPIV5ΔSH-CPI−, or rPIV5ΔSH-Rev may be generated and tested using thesame experimental regiment.

Repeated injections of PIV5 into wild type mice do not cause anynoticeable illness and mice seem to tolerate the injections well.Previous reports indicate that innate immune responses play a criticalrole in controlling PIV5 infection in mouse (He et al., 2002, Virology;303:15-32; and He et al., 2001, J Virol; 75:4068-4079). Because nudemouse, like wild type mouse, has normal innate immune responses, it isexpected that PIV5 will behave similarly in wild type mouse (immunecompetent mouse) to nude mouse in terms of tolerating PIV5 infection aswell as allowing PIV5 replication in cancer cells but restricting PIV5replication in normal cells.

Example 6 Oncolytic Activities of PIV5 Expressing MDA-7/IL-24

MDA-7, also known as IL-24, is a member of IL-10 cytokine family. It hasbeen demonstrated that MDA-7/IL-24 can selectively induce apoptosis inmany human cancer cell lines with minimum damage to normal cells andMDA-7/IL-24 can also inhibit growth of human cancer cell xenografts in anude mouse model system (Nishikawa et al., 2004, Oncogene;23(42):7125-31; Yacoub et al., 2004, Cancer Biol Ther; 3(8):739-51;Nishikawa et al., 2004, Mol Ther; 9:818-828; Gopalkrishnan et al., 2004,Int Immunopharmacol; 4:635-647; and Ramesh et al., 2004, Mol Ther;9:510-518 76-80). Results from phrase I/II clinical trials usingMDA-7/IL-24 indicate MDA-7/IL-24 is safe and effective against solidtumor and melanomas (Introgen Therapeutics, Inc., Austin, Tex.).

The mechanism of selective killing of cancer cells by MDA-7/IL-24 is notclear. It is thought that multiple apoptotic pathways are activated byMDA-7/IL-24 (Wang et al., 2002, J Biol Chem; 277:7341-7347). Atphysiological levels, secreted MDA-7/IL-24 can interact withheterodimers of IL-20R1/R2 or IL-22R1/IL-20R2 and activate STAT-1 andSTAT-3, leading to expression of genes that inhibit uncontrolled cellgrowth. When MDA-7/IL-24 is overexpressed, MDA-7/IL-24 can activateexpression of GADD gene family and lead to apoptosis throughintermediate proteins that are yet identified (Sauane et al., 2004,Cancer Res; 64:2988-2993).

While MDA-7 is known to preferentially kill tumor cells and clinicaltrials using IL-24 as an oncolytic agent have shown promise, thedelivery of IL-24 to the desirable site at sufficient quantity remains achallenge. As described in Example 2, a recombinant PIV5 expressing MDA7(rPIV5-MDA7) has been generated. To generate rPIV5-MDA7, a full-lengthMDA-7/IL-24 gene coding sequence (encoding 206 amino acid residues) thatgives rise to secreted form of MDA-7/IL-24 was inserted into PIV5 genomebetween HN and L gene. This recombinant virus is very effective inkilling tumors in vivo in a mouse model.

In this example, the oncolytic activity of rPIV5-MDA7 will be furthertested. For example, the efficacies of a mutant PIV5 expressing thetumor killing agent MDA-7/IL-24 will be tested in treating metastaticbreast cancer in a nude mouse model as well as in an immune competentmouse model system. In addition, the oncolytic activity of variousmutant PIV5 constructs combined with MDA7 expression will be examined,to further enhance oncolytic activity of the viruses.

Negative strand RNA viruses, such as PIV5, initiate transcription fromthe 3′ end leader sequence, and transcription levels of the viral genesare affected by their distances to the leader sequence. For example, theNP gene of PIV5 which is the closest to the leader sequence is the mostabundantly transcribed, whereas the L gene that is the located mostdistance from the leader sequence is least transcribed. The efficacy ofthe oncolytic virus candidate may be enhanced by increasing theexpression level of the MDA7 protein. To increase the expression levelof the MDA7 gene, the MDA7 gene will be inserted immediately downstreamof the leader sequence and upstream of the NP gene (PIV5-MDA7LN). Therecombinant virus will be recovered and analyzed in vitro the same asPIV5-MDA7. Efficacy of PIV5-MDA7LN will be comparatively studied withthat of PIV5-MDA7 in vitro and in vivo using solid tumor model andmetastatic model in nude mice as well as immune competent model asdescribed in the previous examples. Besides efficacy, thebio-distribution of the virus and tumor biology after virus infectionwill be examined. In addition, the expression of MDA7 in animalsinfected with rPIV5-MDA7 will be examined. In particular, the expressionof MDA7 in tumors infected with rPIV5-MDA7 at 1, 3, 5 and 7 days postinfection will be examined using immunoblotting. The expression of MDA7in sera and internal organs such as lung, kidney and liver will also beexamined.

Expression of MDA7 will be further combined with oncolytic activity ofother mutants (for example, any of those described herein) to furtherimprove efficacy of oncolytic activity of PIV5 as anti-tumor agents. Forinstance, a recombinant virus lacking the conserved C-terminus of V andexpressing MDA7 (rPIV5 AC-MDA7) or containing even more mutations thatare effective in enhancing oncolytic activity of PIV5 will be generated.The virus will be tested in solid tumor model, metastatic cancer modeland LL2/C57/B16 model by examining efficacy, bio-distribution and tumorbiology as described in the previous examples.

Example 7 The Role of AKT in Replication of PIV5

AKT (also known as protein kinase B, PKB) was first discovered inretrovirus AKT8 as a viral proto-oncogene capable of transformingcertain cells (reviewed in Brazil and Hemmings, 2001, Trends BiochemSci; 26:657-664). Identification and cloning of the AKT gene showed thatit has high homology to protein kinases A and C, hence the name PKB.Three mammalian AKT genes (AKT1, 2 and 3, also known as PKBα, β, and γ,respectively) have been identified and they all have serine/threoninekinase activity. v-AKT is the viral form of AKT and is a fusion betweenthe viral Gag and mouse AKT1. All AKT genes contain an N-terminalpleckstrin homology (PH) domain, a catalytic domain, and a C-terminalregulatory domain. There are two major phosphorylation sites within AKT,amino acid residue Thr308 and Ser473, which are phosphorylated by PDK1(PI3K-dependent kinase 1) phosphorylates Thr308 and the rictor-mTORcomplex respectively (Chan et al., 1999, Annu Rev Biochem; 68:965-1014;and Sarbassov et al., 2005, Science; 307:1098-1101). AKT has manydownstream targets, including BAD, CREB, eNOS, I-κB kinase α, GSK-3 andp21 CIP1 (Du and Tsichlis, 2005, Oncogene; 24:7401-7409). AKT is a keyregulator in the PI3K signaling pathway and plays an important role inmany cellular processes such as cell survival, metabolism, growth,proliferation and mobility. AKT has been found to be activated in manycancers, and targeting AKT with small molecules has reduced tumor growthin some circumstances (Redaelli et al., 2006, Mini Rev Med Chem;6:1127-1136; and Yoeli-Lerner and Toker, 2006, Cell Cycle; 5:603-605).It has been reported that AKT1 plays a critical role in breast cancer(Ju et al., 2007, Proc Natl Acad Sci USA; 104:7438-7443; and Sun et al.,2001, Am J Pathol; 159:431-437). While AKT1 and 2 are widely expressedin many organs and cell types, AKT3 is expressed preferentially, but notexclusively in neuronal cells. All three AKT genes share some redundantfunctions, but they all have distinct functions through the studiesusing knockout mice (single AKT knockout: Akt1−/— and Akt2−/− areviable, no report of Akt3 knockout yet) and siRNAs (Stambolic andWoodgett, 2006, Trends Cell Biol; 16:461-466).

Although it has been demonstrated experimentally that anti-tumor virusescan selectively kill tumor cells while causing minimum harm to normalcells, the mechanisms of this selectivity by anti-tumor viruses are notclear.

As shown in Example 2, an AKT inhibitor and siRNA inhibit PIV5 viralprotein expression, the AKT inhibitor inhibits the reporter geneexpression in the mini-genome system, and the AKT inhibitor has noeffect on expression of viral proteins from mammalian gene expressionvector using CMV promoter, indicating AKT likely plays a role inregulating viral RNA synthesis, not viral mRNA translation. Based on theresults that AKT, a kinase that plays a critical role in cancerdevelopment, also plays a critical role in replication of PIV5, it islikely that AKT activation contributes to the selectivity of oncolyticvirus. This example will test the role of AKT in PIV5 replication andthe selectivity of PIV5. Elucidate the role of AKT in replication ofPIV5. siRNA or small molecule inhibitor against AKT1 reduces replicationof PIV5, indicating that AKT plays a critical role in replication ofPIV5. However, the mechanism of regulation of PIV5 replication by AKT isnot clear. AKT possibly plays a critical role in phosphorylation of theP protein of PIV5, which is an essential component of the viral RNApolymerase complex. The role of AKT in phosphorylation of P will beinvestigated and its consequences on virus replication. Understandingthe role of AKT in virus replication will help with designing a moreefficacious oncolytic virus. Preliminary studies indicate AKT, a kinase,plays a critical role in viral RNA synthesis. The role of AKT in virusreplication will be tested. And the contribution of AKT to theselectivity of oncolytic virus (i.e., targeting cancer cellspreferentially and causing minimal damage to normal cells, because ofits dependence on AKT for efficient replication) will be tested. Asdemonstrated in the previous examples, siRNA or small molecule inhibitoragainst AKT1 reduces replication of PIV5, indicating that AKT plays acritical role in replication of PIV5. However, the mechanism ofregulation of PIV5 replication by AKT is not clear. It is likely thatAKT plays a critical role in phosphorylation of the P protein of PIV5,which is an essential component of the viral RNA polymerase complex.

This example will investigate the role of AKT in phosphorylation of Pand its consequences on virus replication. Understanding the role of AKTin virus replication will help with designing a more efficaciousoncolytic virus.

To determine whether AKT plays a role in regulating viral RNA synthesis,cells will be infected with PIV5 at 5 MOIs or mock-infected and at 1 dpithe cells will be treated with actinomycin D (Act D), which inhibitshost RNA synthesis but has no effect on viral RNA synthesis, and AKTinhibitor or DMSO with ³H-UTP for 6 to 8 hours. The cells will be lysedwith 1% SDS, mixed with the same volume of cold trichlotoacetic acid(TCA) (10%) and incubated on ice for 30 minutes. TCA-insolubleprecipitates will be collected on glass filters (GF/C, 2.4-cm; Whatman),washed with 5% cold TCA and ethanol. The radioactivity on the filterswill be measured using a scintillation counter as described in Kim et al(Kim and Kawai, 1998, Biol Pharm Bull; 21:498-505). Total RNA can alsobe extracted from the cells as described before using Qiagen RNApurification kit (Lin et al., 2005, Virology; 338:270-280) and newlysynthesized, 3H-labeled viral RNA will be measured using a scintillationcounter. To further determine the effect of AKT inhibitor on RNAsynthesis (transcription vs. replication), the cells labeled with 3H-UTPas described before will be lysed with a mild lysis buffer containing0.5% Triton X-100 and 50 mM Tris-Cl (pH 7.4) (not to disrupt RNP,NP-encapsidated RNA structure). Half of the lysis will be treated withRNase A, which degrades viral mRNA as well as host RNA but not viralgenome RNA since Npencapsidated RNA genome is resistant to RNase Atreatment, the other half will be used as control. The radioactivitywill be measured with a scintillation counter. Alternatively, viralgenomic RNA will be obtained as described in Lin et al usingimmuno-precipitation with antibody against NP (Lin et al., 2005,Virology; 338:270-280) or from pellet fraction as described in Randallet al. (Randall and Bermingham, 1996, Virology; 224:121-129). Ingeneral, viral mRNA contains polyA tail and can be purified usingpolydT; whereas viral genome RNA that is encapsidated can be purifiedaway from other RNA by anti-NP antibody or by centrifugation inultra-centrifuge because of RNP (genome RNA and NP) can be pelleted dueto its weight and density.

If AKT affects viral RNA replication, it may result in reduced viralmRNA expression due to the reduced amount of viral RNA genome templateavailable for transcription. To measure the role of AKT on viraltranscription directly, the minigenome system with a defectiveanti-genome promoter may be used. In this system, only viral RNAtranscription will be measured since no viral RNA replication can occurdue to the mutation in the anti-genomic promoter.

To determine whether AKT plays a role in termination/reinitiation duringviral RNA synthesis, a mini-genome with two reporter genes has beenconstructed and will be tested. If the effect of AKT inhibitor islimited to initiation, the ratio of activities from two reporter geneswill be similar between DMSO and AKT inhibitor treated cells, eventhough overall reporter gene activities of AKT inhibitor-treated cellswould be lower than that of DMSO-treat cells. If AKT has a role intermination/reinitiation of viral mRNA transcription, the ratios ofexpression levels of two reporter genes will be very different betweenDMSO and AKT inhibitor treated cells. If AKT plays a role inelongation/processivity, ratios of R-Luc to F-Luc from the mini-genomesystem will be different between DMSO and AKT inhibitors because thesecond reporter gene in the mini-genome will be transcribed less due tolower processivity in the presence of AKT inhibitors.

Effects of AKT inhibitors on transcription termination and reinitiationcan also be examined in PIV5-infected cells using quantitative real timePCR (qRT-PCR). If AKT plays a role in RNA replication, effects of AKTinhibitors on synthesis of vRNA (genomic RNA) or cRNA (anti-genomicsense RNA) can be detected with qRTPCR by measuring relative amount ofvRNA and cRNA within NP-encapsidated genome RNA. Also, effects of AKTinhibitors on initiation of replication vs. elongation of replicationcan also be examined using qRT-PCR.

If a reduced amount of genome RNA is detected in the presence of AKTinhibitor, it may indicate that AKT plays a role in synthesis of viralgenome RNA (replication). It is possible that inhibition of AKT mayresult in reduced viral RNA replication but increased viral mRNAsynthesis. In this case, increased ratio of viral mRNA vs. viral RNA(vRNA or cRNA) may be observed. In case AKT affects initiation of viralRNA replication or transcription, more RNAs close to leader and trailersequences may be detected.

Phosphorylation Status of AKT, NP, P and L. AKT inhibitors known toinhibit AKT phosphorylation such as the AKTIV inhibitor inhibited viralRNA synthesis (Kau et al., 2003, Cancer Cell; 4:463-476), indicatingphosphorylation of AKT is important for its role in viral RNA synthesis.However, since some of the inhibitors can prevent phosphorylation of AKTby preventing conformation change associated with phosphorylation, it ispossible that phosphorylation per se is not required for AKT's role inviral RNA synthesis. To determine the role of phosphorylation of AKT inviral RNA synthesis, phosphorylation status of AKT will be determined inmock and PIV5-infected cells. To determine whether phosphorylation ofAKT is required for its role in viral RNA synthesis, siRNA targetingendogenous AKT 1 will be used. The cells will then be supplemented witha kinase inactive mutant AKT (Thr308Ala or Ser374Ala or both) or wildtype AKT that contain changed nucleotide coding sequences withoutchanging amino acid sequences to be resistant to the siRNA from anexpression vector. If kinase inactive mutants can rescue the reductionof PIV5 replication caused by siRNA against endogenous AKT, it wouldindicate kinase activity of AKT is not required for its function inviral RNA synthesis. In addition, the effect of a dominant negativemutant of AKT 1 (AKT1DN) (van Weeren et al., 1998, J Biol Chem;273:13150-13156) will be tested in PIV5-infected cells as well as in themini-genome system.

In PIV5 infected cells, P is phosphorylated. In paramyxovirus Sendaivirus, NP is phosphorylated as well (Lamb and Choppin, 1977, Virology;81:382-397). Phosphorylation status of L is not clear. To determine theeffect of AKT on their phosphorylation, phosphorylation status of NP, Pand L in the absence as well as in the presence of AKT inhibitor will beexamined. The cells will be infected with PIV5 at 5 MOIs or mockinfected. At 1 dpi, the cells will be treated with DMSO or AKT inhibitorand labeled with ³³P-phosphor or ³⁵S-Met and ³⁵S-cys for 6 to 8 hours.The cells will be then precipitated with antibodies against NP, P, L orAKT. Total amount of NP, P, L and AKT synthesized during the time periodwill be indicated by amount of ³⁵S-labeled protein and amount ofphosphorylated form of NP, P, L and AKT will be indicated by amount of³³P-labeled NP, P, L and AKT. It is possible that phosphorylation statusof P changes over the course of infection. To examine thephosphorylation of NP, P, L and AKT over time, a time course of theexperiment will be carried out. To directly measure the effect of AKT onphosphorylation of NP, P and L, a plasmid encoding NP, P or L will betransfected alone or with a plasmid encoding AKT 1. The cells will thenbe labeled with ³³P-orthophosphor or ³⁵S-Met and ³⁵S-Cys as well astreated with AKT inhibitor or DMSO as before. Phosphorylated form andtotal amount of NP, P, L and AKT will be determined usingimmunoprecipitation with antibodies against NP, P, L or AKT.

Mapping the AKT phosphorylation site within P of PIV5 and determine therole of phosphorylation of P by AKT in viral RNA synthesis. In theprevious examples it was shown that the P protein is phosphorylated ininfected cells and AKT inhibitor can inhibit this phosphorylation.Importantly, AKT phosphorylates recombinant P purified from bacteria.However, exact phosphorylation sites of P of PIV5 are not clear. Tostudy the impact of AKT mediated phosphorylation of the P protein by amore direct approach, phosphorylation sites of AKT within the P proteinwill be determined and then the effects of mutating these sites will beexamined. P is highly enriched in threonine and serine residues (36 Serand 32 Thr). To determine the sites, the P protein will be purified frominfected cells. Purified P will be subjected to mass spectrometry (MS)analysis and phosphorylation sites will thus be determined. In case MSis not sufficient to pinpoint the sites for phosphorylation, mutationsof suspected sites will be generated and tested. For instance, if atrypsin-digested peptide of P is identified as phosphorylated and thepeptide happens to have more than one Ser or Thr residues, the Ser orThr residues will be mutated to Ala individually, the mutant P proteinswill be purified and subjected to the same MS analysis if a direct³³P-orthophosphor-labeling experiment is not sufficient to detect thedifferences of phosphorylation between wild type and mutant P proteins.In addition, a mutational analysis of the P protein will be carried outusing bacteria purified protein with in vitro kinase assay by analyzingphosphorylation of P with deletion as well as point mutation todetermine the phosphorylation site of AKT within P.

If the AKT phosphorylation sites of P are determined, mutationsaffecting phosphorylation of P will be generated and the effect of AKTinhibitor on phosphorylation status will be examined. If AKT affects thephosphorylation of P, AKT inhibitors would inhibit phosphorylation ofthe sites in vitro kinase assay. To determine the importance of thesephosphorylation sites in viral RNA synthesis, the sites will be mutatedand the effect of the mutations will be examined using the mini-genomesystem with functional leader and trailer sequences, which RNAtranscription and replication can be tested as, and the mini-genomesystem with the defective anti-genome promoter, which only viraltranscription can be examined as previously described (Lin et al., 2005,Virology; 338:270-280). Further, the effect of the mutations will beexamined using reverse genetics system, if the mutations are not lethal.A similar approach will be followed to determine phosphorylation sitesof V, NP and L and the roles of their phosphorylation in viral RNAsynthesis if they are phosphorylated.

Ongoing experiments on determining phosphorylation sites of P have beenproductive; one phosphorylation site has been identified and anothersite has been mapped to a small peptide with two serine residues.Determining phosphorylation sites and their function is a challengingtask. Such studies will focus first on the P protein. If phosphorylationof P is not important for its function, an unlikely possibility, focuswill shift to the NP protein. Finally the L protein will be studied.Because of its size of L, it may be necessary to break the L proteindown into different domains for analysis. The L protein may be expressedas two proteins: one containing domains 1, 2 and 3 (L123); and the othercontaining domains 4, 5 and 6 (L456). Whether L123 or L456 isphosphorylated and how AKT affects its phosphorylation will bedetermined as described above. L123 or L456 can be further reduced tosmall protein such as L12, which only contains domain 1 and 2. L islikely organized as 6 domains. Alternatively, to determine thephosphorylation sites using MS, purified virions can be used to obtainsufficient amount of viral proteins.

Examine the interactions between AKT and NP, P or L. It is possible thatAKT affects phosphorylation of P or NP or L through an intermediate hostfactor in infected cells. It is also possible that AKT interacts withNP, P or L directly. To examine whether AKT interacts with viral RNAsynthesis machinery, NP, P or L directly, coimmunoprecipitation (co-IP)will be carried out in infected cells as well as in cells transfectedwith AKT plus NP, P or L as previously described. In addition, theinteractions will be detected in the presence of AKT inhibitors toinvestigate the effects of inhibitor on AKT and its interactingpartners. To detect weak interaction, cross-linking reagents will beutilized. If interactions are detected between AKT1 and NP, P or L intransfected cells, whether AKT interacts directly with NP, P or L willbe examined using purified proteins for determining direct interactionbetween V and AKT1.

Co-IP using infected cells provides knowledge in real infection and theresult is preferred. However, because NP interacts with P and Pinteracts with L, it may not be possible to dissect interactions betweenAKT and any of the NP, P or L protein in infected cells withoutinterference from each other. Co-IP of transfected cells thus provides aclear means to determine whether AKT can interact with NP, P or Lwithout other viral protein. Whether addition of any component willfurther enhance the interaction will be examined if it is found one ofthem (NP, P or L) interacts with AKT. For instance, if AKT interactswith P, NP or L or both will be added to the transfection mixture to seewhether there is enhancement of the interaction between P and AKT. Inpreliminary studies, AKT phosphorylates recombinant P protein purifiedfrom bacteria, indicating AKT can interact directly with P that ispurified from bacteria. Thus, the detection of a P and AKT interactionin infected mammalian cells is expected.

The role of activated AKT in efficient virus replication and oncolyticactivity of PIV5 without AKT phosphorylation site. AKT plays anessential role in efficient virus replication in cancer cells such asHeLa. In addition, the virus replicates better in cancer cells thannormal cells in vivo. It is not clear whether an overactive AKT issufficient to convert non-hospitable normal cells into susceptiblecells. To test this, an active AKT will be expressed in normal cells andexamine virus replication in the newly established cells in vivo.Towards this end, a lentiviral vector will be generated expressing abicistronic mRNA containing a constitutively active AKT (T308E andS473D) and a GFP that are separated by an internal ribosome entry site(IRES) from EMCV (EIRES). The hTERT cells (normal HMEC immortalized withhTERT) will be transduced with the vector (a lentiviral vector with onlyEIRES and GFP will be used as a control) and the transduced cells willbe sorted by the expression of GFP. The sorted cells will then beinjected into nude mice.

In addition, MDA-MB-435 cells will be injected into nude mice as well(total three kinds of cells in equal numbers will be injected into eachnude mouse at different locations). The cells will be infected withrPIV5-RL in situ and replication of rPIV5-RL will be monitored using anIVIS camera. If AKT phosphorylation site within P is determined asdescribed above, a recombinant PIV5 containing a defective AKTphosphorylation site will be generated. The efficacy of this virus inreducing tumor growth will be examined in a similar fashion.

Consistent with published reports indicating that AKT is activated inMDA-MB-435 cells, the previous examples compared levels ofphosphorylation of AKT in the hTERT and MDAMB-435 cell and found higherlevels of AKT phosphorylation in MDA-MB-435 cells than that in the hTertcells. If activated AKT is sufficient for efficient virus replication,renilla luciferase activities from the hTERT cells with constitutivelyactive AKT will be higher than the hTERT cells with only GFP in vivo.However, if renilla luciferase activities from the hTERT cells withconstitutively active AKT are similar to that of the hTERT cells withcontrol (GFP only), it indicates that active AKT alone is not sufficientfor efficient virus replication. Since it is known that efficient virusreplication requires many host factors, expressing only active AKT innormal cells such as the hTERT cells may not be sufficient to convertnon-hospitable normal cells into susceptible cells. For instance, arobust interferon signaling system in normal cells such as hTERT cellsmay prevent efficient virus replication even in the presence ofactivated AKT. Since AKT plays a critical role in virus replication,recombinant PIV5 lacking AKT phosphorylation site is likely to bedefective in virus replication. If this virus has oncolytic activity, itmay be less selective since it may not prefer cancer cells which oftenhave activated AKT. Because the virus is likely to be defective in virusreplication, it is likely that it is not as effective as wild type inreducing tumor growth. The understanding of the role of AKT in virusreplication gained from this example will assist in the design of moreefficacious oncolytic viruses.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

What is claimed is:
 1. A method of treating a subject with a cancer, themethod comprising administering to the subject an effective amount of acomposition comprising an isolated recombinant parainfluenza virus 5(PIV5), wherein the PIV5 comprises one or more mutations.
 2. The methodof claim 1, wherein the subject is a companion animal.
 3. The method ofclaim 2, wherein the companion animal is a dog.
 4. A method of imaging atumor in a subject, the method comprising administering to the subject arecombinant parainfluenza virus 5 (PIV5) expressing a fluorescentpolypeptide or detectable agent.
 5. The method of claim 1 wherein thetumor is a primary tumor and/or a metastatic tumor.
 6. The method ofclaim 1, wherein the tumor is selected from the group consisting ofmelanoma, basal cell carcinoma, colorectal cancer, pancreatic cancer,breast cancer, prostate cancer, lung cancer (including small-cell lungcarcinoma and non-small-cell lung carcinoma, leukemia, lymphoma,sarcoma, ovarian cancer, Kaposi's sarcoma, Hodgkin's lymphoma,Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, head andneck cancers, malignant pancreatic insulanoma, malignant carcinoid,urinary bladder cancer, premalignant skin lesions, testicular cancer,lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, cervical cancer,kidney cancer, endometrial cancer, glioblastoma, and adrenal corticalcancer.
 7. The method of claim 1, wherein administration of PIV5 isintratumoral, subcutaneous, intravenous, intranasal, intraperitoneal,intracranial, oral, or in situ.
 8. The method of claim 1 furthercomprising administration of an additional therapeutic agent.
 9. Themethod of claim 1, wherein a mutation comprises a mutation of the V/Pgene, a mutation of the shared N-terminus of the V and P proteins, amutation of residues 26, 32, 33, 50, 102, and/or 157 of the sharedN-terminus of the V and P proteins, a mutation lacking the C-terminus ofthe V protein, a mutation lacking the small hydrophobic (SH) protein, amutation of the fusion (F) protein, a mutation of the phosphoprotein(P), a mutation of the large RNA polymerase (L) protein, a mutationincorporating residues from canine parainfluenza virus, and/or amutation that enhances synctial formation.
 10. The method of claim 1,wherein a mutation is selected from the group consisting ofrPIV5-V/P-CPI−, rPIV5-CPI−, rPIV5-CPI+, rPIV5VΔC, rPIV-Rev, rPIV5-RL,rPIV5-P-S157A, rPIV5-P-S308A, rPIV5-L-A1981D and rPIV5-F-S443P,rPIV5-MDA7, rPIV5ΔSH-CPI−, or rPIV5ΔSH-Rev, and combinations thereof.11. The method of claim 1, wherein the PIV5 further comprises nucleotidesequences encoding a tumor killing heterologous polypeptide and/orheterologous RNA.
 12. The method of claim 11, wherein the heterologouspolypeptide comprises MDA7.
 13. An oncolytic agent comprising a mutantparainfluenza virus 5 (PIV5) comprising one or more the mutationsselected from the group consisting of a mutation of the V/P gene, amutation of the shared N-terminus of the V and P proteins, a mutation ofresidues 26, 32, 33, 50, 102, and/or 157 of the shared N-terminus of theV and P proteins, a mutation lacking the C-terminus of the V protein, amutation lacking the small hydrophobic (SH) protein, a mutation of thefusion (F) protein, a mutation of the phosphoprotein (P), a mutation ofthe large RNA polymerase (L) protein, a mutation incorporating residuesfrom canine parainfluenza virus, and/or a mutation that enhancessynctial formation.
 14. The oncolytic agent of claim 13, wherein amutation is selected from the group consisting of rPIV5-V/P-CPI−,rPIV5-CPI−, rPIV5-CPI+, rPIV5VΔC, rPIV-Rev, rPIV5-RL, rPIV5-P-S157A,rPIV5-P-S308A, rPIV5-L-A1981D and rPIV5-F-S443P, rPIV5-MDA7,rPIV5ΔSH-CPI, rPIV5ΔSH-Rev, and combinations thereof.
 15. The oncolyticagent of claim 13, wherein the mutant PIV5 further comprises nucleotidesequences encoding a tumor killing polypeptide or RNA.
 16. The oncolyticagent of claim 15, wherein the heterologous polypeptide comprises MDA7.17. A composition comprising an oncolytic agent of claim 13 and apharmaceutically acceptable carrier.